Protection of Progenitor Cells and Regulation of Their Differentiation

ABSTRACT

The present invention relates to the use of polysulfated polysaccharides in combination with progenitor cells to improve the viability of the progenitor cells including improving the cryopreservation of the progenitor cells and provides novel compositions, methods and uses. The present invention also relates to the use of polysulfated polysaccharides to regulate the proliferation and differentiation of progenitor cells.

FIELD OF THE INVENTION

The present invention relates to the use of polysulfated polysaccharidesin combination with progenitor cells to improve the viability of theprogenitor cells including improving the cryopreservation of theprogenitor cells and provides novel compositions, methods and uses. Thepresent invention also relates to the use of polysulfatedpolysaccharides to regulate the proliferation and differentiation ofprogenitor cells.

BACKGROUND OF THE INVENTION

Human progenitor cells are the immature cells that give rise to all ofthe different types of mature cells that make up the organs and tissuesof the adult body. The transition from progenitor cell to mature,specialised adult cell is via a process called differentiation.

Progenitor cells in the body take different pathways of differentiationin response to different stimuli from their environment. Similarly,progenitor cells in the laboratory can be stimulated to differentiatealong different pathways by exposing them to various combinations ofbiochemicals. With appropriate stimuli, progenitor cells candifferentiate into, among other tissues, blood cells, bone, cartilage,fat, blood vessels or heart muscle. Because of this, great interest isgiven to the use of progenitor cells as the basis of treatments torepair and re-grow of a range of tissues and organs.

Progenitor cells exist in the embryo and also in adult tissues such asbone marrow, fat, skin and dental pulp, though in much smaller relativenumbers than in the embryo. The two types of adult progenitor cells arehaematopoietic, which give rise to new bone marrow and blood cells, andnon-haematopoietic, which give rise to solid organs and tissues, such asbone, heart and cartilage. Haematopoietic-type adult progenitor cellscan be readily obtained from bone marrow and are already being usedclinically. However, technology related to non-haematopoietic-type adultprogenitor cells is much less developed due to the difficulty ofobtaining sufficient numbers of these cells and of growing them in thelaboratory.

In order to use progenitor cells in therapy it is necessary to be ableto successfully store the progenitor cells prior to their use. Theprogenitor cells must be stored in such a way that they are effectivelypreserved and their viability is maintained. In general, the progenitorcells are cryopreserved for storage and thawed prior to use.

Cryogenic preservation (storage below −100° C.) of cell cultures iswidely used to maintain backups or reserves of cells without theassociated effort and expense of feeding and caring for them. Thesuccess of the freezing process depends on four critical areas, properhandling of the cultures, controlled freezing, proper storage and anappropriate cryoprotective agent. The last point is particularlyimportant and a suitable agent can assist in maintaining the viabilityof the cells.

In a clinical setting, it is particularly important that followingcryopreservation, the cells remain viable and any increase in theviability of the cells will boost the effect of the treatment.

In addition, in order for the progenitor cells to be therapeuticallyeffective it is necessary for them to differentiate into the requiredcell type. Thus, there is also a need to develop effective regulators ofprogenitor cell differentiation to ensure that the progenitor cellsdifferentiate into the required cell type.

Furthermore, there is also a need to develop effective regulators ofprogenitor cell proliferation. It is often desirable for the progenitorcells to proliferate both in vitro and in vivo.

Therefore, there remains a need for agent(s) which can protect theprogenitor cells during cryopreservation, enhance their viability,regulate their differentiation and/or regulate their proliferation.

SUMMARY OF THE INVENTION

The present inventors have now found that polysulfated polysaccharidesor biologically active molecular fragments thereof can improve theviability of progenitor cells. In particular, present inventors havefound that polysulfated polysaccharides or biologically active molecularfragments thereof can enhance cryopreservation of progenitor cells.

The present inventors have also found that polysulfated polysaccharidesor biologically active molecular fragments thereof can regulate theproliferation of progenitor cells.

The present inventor has also found that polysulfated polysaccharides orbiologically active molecular fragments thereof can regulate thedifferentiation of progenitor cells. Regulation may be upregulation ordownregulation. It has been found that polysulfated polysaccharides orbiologically active molecular fragments thereof can regulatedifferentiation into chondrocytes, osteoblasts, and adipocytes. Inparticular, it has been found that polysulfated polysaccharides orbiologically active molecular fragments thereof can inducechondrogenesis.

These findings indicate that polysulfated polysaccharides orbiologically active molecular fragments thereof can be used incombination with progenitor cells to improve or enhance the viability ofthe progenitor cells after cryopreservation and can be used incombination with progenitor cells in in vitro and in vivo methods anduses.

These unexpected findings therefore open up the possibility of using thepolysulfated polysaccharides or a biologically active molecular fragmentthereof in a number of new applications. For example, by regulatingdifferentiation, particularly chondrogenesis, it is possible, amongother things, to rebuild cartilage and intervertebral discs, prevent thedegradation of joints and enhance the repair of avascular connectivetissues. Prior to the present invention, it was not known that thepolysulfated polysaccharides could regulate differentiation ofprogenitor cells, particularly chondrogenesis. Furthermore, byregulating proliferation, it is possible to control the production ofprogenitor cells both in vitro and in vivo. While the use ofpolysulfated polysaccharides in relation to osteo arthritis (OA)treatments per se has been published, it was not previously known thatpolysulfated polysaccharide have advantageous cryopreservationproperties in relation to progenitor cells or that they can regulatedifferentiation and/or cell proliferation of said cells. Therefore, thisopens up new treatment avenues that were not considered before.

As used herein a “biologically active molecular fragment” is a portionof a molecule of the invention which maintains a defined activity of thefull-length molecule, namely in one embodiment to be able to enhanceviability, to regulate cell differentiation and/or to regulate cellproliferation.

Accordingly, in a first embodiment, the present invention provides acomposition comprising a progenitor cell together with a polysulfatedpolysaccharide or biologically active molecular fragment thereof.

In a further embodiment, the present invention provides a compositioncomprising progenitor cells, and a polysulfated polysaccharide orbiologically active molecular fragment thereof, together with a carriermedium.

The carrier medium may be a culture medium, bioscaffold,cryopreservation medium, physiological media and/or a pharmaceuticallyacceptable carrier.

In a further embodiment, the present invention provides a compositioncomprising progenitor cells and a polysulfated polysaccharide orbiologically active molecular fragment thereof, together with acryopreservation medium.

The composition may be used both in vitro and in vivo.

The composition can contain any number of progenitor cells. In a furtherembodiment, the present invention contains about 1000 to about 1×10¹⁰progenitor cells. In a further embodiment, the present inventioncontains about 1×10⁵-1×10⁹ cells. In a further embodiment, the presentinvention contains 100,000 to about 5×10⁸progenitor cells. In a furtherembodiment, the present invention contains about 500,000 to about 2×10⁸progenitor cells, about 1×10⁶ to about 2×10⁸ progenitor cells, or about1×10⁶ to about 1×10⁸ progenitor cells. In a yet further embodiment, thecomposition contains about 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶,7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷,8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, or9×10⁸ progenitor cells. In yet a further embodiment, the compositioncontains about 1×10⁸ progenitor cells.

In one embodiment, the concentration of the polysulfated polysaccharidein the composition will depend on the number of cells in thecomposition. Thus, in one embodiment, the concentration of thepolysulfated polysaccharide in the composition is from 500 ng/ml/millioncells-10 mg/ml/million cells, or 500 ng/ml/million cells-2000μg/ml/million cells, 1 μg/ml/million cells-1000 μg/ml/million cells, or1 μg/ml/million cells-500 μg/ml/million cells.

In a further embodiment, the polysulfated polysaccharide concentrationis in the range of 500 ng-10 μg/ml/million cells; 1-10 μg/ml/millioncells; 1 μg-8 μg/ml/million cells; 1 μg-6 μg/ml/million cells; 1-5μg/ml/million cells; 1-3 μg/ml/million cells; 2 μg-6 μg/ml/millioncells; 2.5 μg-5 μg/ml/million cells; or 3 μg-5 μg/ml/million cells. In afurther embodiment, the polysulfated polysaccharide concentration is inthe range of 1 μg-100 μg/ml/million cells; 1 μg-50 μg/ml/million cells;1 μg -20 μg/ml/million cells; 1 μg-15 μg/ml/million cells; 10 μg-100μg/ml/million cells; 20 μg-100 μg/ml/million cells; or 50 μg-100μg/ml/million cells. In a further embodiment, the polysulfatedpolysaccharide concentration is in the range of 1 μg-1000 μg/ml/millioncells; 100 μg-800 μg/ml/million cells; 100 μg-600 μg/ml/million cells;100 μg-500 μg/ml/million cells; 200 μg-500 μg/ml/million cells. In afurther embodiment the polysulfated polysaccharide concentration is inthe range of 250 μg-500 μg/ml/million cells.

In one embodiment, the polysulfated polysaccharide concentrationcomprises 500 ng, 1 μg, 2 μg, 2.5 μg, 5 μg, 10 μg, 15 μg, 20 μg, 30 μg,40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200 μg, 250μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, 1000 μg, 1050 μg, 1100 μg,1150 μg, 1200 μg, 1250 μg, 1300 μg, 1350 μg, 1400 μg, 1450 μg, 1500 μg,1550 μg, 1600 μg, 1650 μg, 1700 μg, 1750 μg, 1800 μg, 1850 μg, 1900 μg,1950 μg, or 2000 μg/ml/million cells. In a further embodiment, thepolysulfated polysaccharide concentration comprises polysulfatedpolysaccharide concentrations comprise 200 μg/ml/million cells, 250μg/ml/million cells, 300 μg/ml/million cells, 400 μg/ml/million cells,or 500 μg/ml/million cells. In a yet further embodiment, thepolysulfated polysaccharide concentration comprises 250 μg/ml/millioncells or 500 μg/ml/million cells.

Alternatively, the concentration of the polysulfated polysaccharide isindependent of the number of cells in the composition. Thus in a furtherembodiment of the present invention the concentration of thepolysulfated polysaccharide in the composition is from 500 ng/ml-10mg/ml; 500 ng/ml-2000 μg/ml; 1 μg/ml-1000 μg/ml; or 1 μg/ml-500 μg/ml.

In a further embodiment, the polysulfated polysaccharide concentrationis in the range of 500 ng-10 μg/ml; 1 μg-10 μg/ml; 1 μg-8 μg/ml; 1 μg-6μg/ml; 1 μg-5 μg/ml; 1 μg-3 μg/ml; 2 μg-6 μg/ml; 2.5 μg-5 μg/ml; or 3μg-5 μg/ml. In a further embodiment, the polysulfated polysaccharideconcentration is in the range of 1 μg-100 μg/ml; 1 μg-50 μg/ml; 1 μg-20μg/ml; 1 μg-15 μg/ml; 10 μg-100 μg/ml; 20 μg-100 μg/ml; or 50 μg-100μg/ml. In a further embodiment, the polysulfated polysaccharideconcentration is in the range of 1 μg-1000 μg/ml; 100 μg-800 μg/ml; 100μg-600 μg/ml; 100 μg-500 μg/ml; 200 μg-500 μg/ml. In a furtherembodiment, the polysulfated polysaccharide concentration is in therange of 1 mg-1000 μg/ml; 100 μg-800 μg/ml; 100 μg-600 μg/ml; 100 μg-500μg/ml; 200 μg-500 μg/ml. In a further embodiment, the polysulfatedpolysaccharide concentration is in the range of 250 μg-500 μg/ml.

Further polysulfated polysaccharide concentration comprises 500 ng, 1μg, 2 μg, 2.5 μg, 5 μg, 10 μg, 15 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg,70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850μg, 900 μg, 950 μg, 1000 μg, 1050 μg, 1100 μg, 1150 μg, 1200 μg, 1250μg, 1300 μg, 1350 μg, 1400 μg, 1450 μg, 1500 μg, 1550 μg, 1600 μg, 1650μg, 1700 μg, 1750 μg, 1800 μg, 1850 μg, 1900 μg, 1950 μg, or 2000 μg/ml.Further polysulfated polysaccharide concentrations comprise 200 μg/ml,250 μg/ml, 300 μg/ml, 400 μg/ml, or 500 μg/ml. Further polysulfatedpolysaccharide concentrations comprise 250 μg/ml and 500μg/ml.

Further compositions contain a total polysulfated polysaccharide contentof 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 15 mg,20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 70 mg, 80mg, 90 mg, or 100 mg. Further compositions contain a total polysulfatedpolysaccharide content of 15-70 mg, 20-60 mg, or 25-50 mg.

A further embodiment comprises about 1×10⁶-1×10⁸ progenitor cells and25-50 mg polysulfated polysaccharide. A further embodiment contains1×10⁸ progenitor cells and 25-50 mg/ml polysulfated polysaccharide.

In a further embodiment, the polysulfated polysaccharide may beadministered in an amount such as to produce a concentration of thepolysulfated polysaccharide in the biological compartment of 0.01 to 100micrograms/ml biological media, for example 5 0.1 to 50 micrograms perml biological media, 0.1 to 50 micrograms per ml biological media, 0.1to 10 micrograms per ml biological media, 1 to 10 micrograms per mlbiological media, 2 to 8 micrograms per ml biological media, 4 to 6micrograms per ml biological media, or 4, 5, or 6 micrograms per mlbiological media.

By biological compartment, it is meant an area of the body such as theintervertebral disk, muscle, synovial joints, intra synovial tissue(meniscus, synovium), extra synovial tissue (capsule), intra tendon,extra tendon, cardium, pericardium, cardiac muscle, and/or intra adiposetissue, intra-ligamentum, extra-ligamentum, intra-dermal, subdermal,intra-peritoneally, intra-venously, intra-arterally. The biologicalmedia will depend on the biological compartment. Biological mediaincludes blood, serum, plasma, synovial fluid, peritoneal fluid, serousfluid, or adipose tissues. Thus, for example, in a further embodiment,the polysulfated polysaccharide may be administered in an amount such asto produce a concentration of the polysulfated polysaccharide in thesynovial joints of 1 to 10 micrograms per ml synovial fluid.

Carrier Medium

The composition may contain a carrier medium. In one embodiment, thecarrier medium is an aqueous solution. The medium may optionally containfurther components which preserves the normal physiological structureand functions of the cells, particularly in relation to maintainingtheir environmental osmolarity, pH, integrity and fluidity of its plasmamembrane and intra-cellular organelles.

Suitable carriers for this invention include those conventionally usedalone and in combination, e.g., water, saline, aqueous dextrose,lactose, Ringer's solution, Krebs mammalian Ringer solution, Earles'ssolution, Gey's solution, Simm's solution, Tyrode solution, hyaluronan,physiological buffered saline (PBS), Locke's solution, Hank's solution,Clark and Lubs buffer, buffers; buffers composed of MES-NaOH,HEPES-NaOH, TRICINE-NaOH, EPPS-NaOH, BICINE-NaOH,Tris(hydroxymethyl)aminomethane-HCl, Glycine-NaOH, sodiumbicarbonte-CO₂, sodium carbonate-bicarbonate, sodium cacodylate, sodiumhydrogen maleate-NaOH; culture media such as, Eagle's medium, Dulbecco'smedium or buffer, McCoy's medium, Click's medium, Ames' medium, alphaMEM, DMEM, Ham's F12, Ham's F10, RPMI-1640CMRL 1066, and 1415 NCTC 135;commercial specialist cell line media eg Stemline® and Megacell® orcommercial cryopreservation agents such as Profreeze® and CryoStor®.

Thus, in one embodiment, the carrier medium is an aqueous medium whichmay optionally further include one or more of the following components:

-   -   organic and/or inorganic salts;    -   buffers    -   proteins such as BSA or transferin;    -   growth factors and cytokines, including insulin like growth        factor, insulin, fibroblast like growth factors; BMP-TGF-beta        super family (eg, BMP-2, BMP-7, BMP-8, TGF beta) and fibroblast        growth factor family, IGF, FGF, EGF, PDGF, VEGF;    -   animal sera including FBS, new-born calf, all other mammalian        species;    -   cryopreservation agents such as Profreeze® and CryoStor®;    -   cryoprotectorants, including dimethyl sulfoxide (DMSO),        glycerol, trehalose, sucrose and other sugars or        dimethylacetamide;    -   carbohydrates;    -   vitamins/co-factors;    -   hormones    -   antibiotics    -   attachment factors;    -   amino acids;    -   plasma expanders like dextran;    -   plasma both human and other mammalian species;    -   plasma substitute;    -   hyaluronan and/or hyaluronic acid, both natural or cross linked.

Thus, in one embodiment, the carrier medium comprises an aqueous mediaselected from water, saline, aqueous dextrose, lactose, a bufferedsolution, hyaluronan and glycols, physiological buffered saline (PBS),Ringer's solution, Locke's solution, Hank's solution, minimum essentialmedium, minimum essential medium alpha (alpha MEM), or DMEM. In oneembodiment, the carrier medium comprises alpha MEM. In an alternativeembodiment, the carrier medium comprises DMEM. In an alternativeembodiment, the carrier medium comprises HAMS 12.

The carrier medium may additionally comprise cryopreservation agentssuch as propriety preparations like Profreeze® or CryoStor®.

In an alternative embodiment, the carrier medium may comprisecryopreservation agents such as propriety preparations like Profreeze®or CryoStor® as the aqueous solution. In this embodiment, thecomposition does not contain carriers like water, saline, aqueousdextrose, lactose, Ringer's solution, a buffered solution, hyaluronan,physiological buffered saline (PBS), Locke's solution, Hank's solution,alpha MEM; DMEM; or HAMS F12 and instead comprises a cryopreservationagents such as propriety preparations like Profreeze® or CryoStor®,optionally in combination with one or more cryoprotectorants such asdimethyl sulfoxide (DMSO), glycerol, trehalose, sucrose and other sugarsor dimethylacetamide. As an example, the carrier medium may comprise orconsist of Profreeze® and DMSO.

The carrier medium may act as a culture medium and may be supplementedwith organic and/or inorganic salts, carbohydrates, vitamins, aminoacids and/or other entities which fulfil the nutritional requirements ofthe cell allowing them to divide and function normally in vitro. In oneembodiment, the carrier medium further comprises serum and/or proteinsupplements. Thus, the culture media may be supplemented with proteins,including but not limited to BSA or transferin. In addition to, orinstead of, the culture medium may be supplemented with serum, forexample foetal or neonatal blood which contain growth factors, eg foetalcalf serum. Recipes for the preparation and method of use of these mediaare well known to those skilled in the art. Suitable media can be foundin Adams R L P. Cell culture for Biochemists. Elsevier/North-HollandBiomedical Press, Amsterdam, New York, Oxford. 1980, pp 84-97 and pp246-260. (ISBN 0-444-80199-5), and Dawson R M C, Elliott D C, Elliott WH and Jones K M, Data for Biochemical Research (Third Edition),Clarendon Press, Oxford, 2002, pp 417-448 (ISBN 0-19-855299-8) thecontents of which are incorporated in its entirety.

In one embodiment, the carrier medium acts as a cryopreservation medium.Cryopreservation media for freeze-thawing cells includes the use of thecommonly known carriers and/or culture media including the aqueous mediadescribed herein (either alone or in combination with serum proteinsupplements). Alternatively, the carrier medium may comprise or includepropriety preparations such as Profreeze® and CryoStor®. To function asa carrier medium, in general compounds are added which protect the cellmembrane and organelles from damage by ice crystals formed during thefreeze-thawing process.

Thus, in a further embodiment, the carrier medium further comprises oneor more cryoprotectorants. Suitable agents or cryoprotectorants includedimethyl sulfoxide (DMSO), glycerol, sucrose and other sugars. Examplesof suitable agents can be found in Brudder S, Jaiswal N, Hainsworth S,WO9739104; Farrant, J. 1980. General observations on cell preservation.In: M. J. Ashwood-Smith and J. Farrant, Eds. Low TemperaturePreservation in Medicine and Biology, Pitman Medical Limited, Kent,England, p. 1-18; Frederick V, et al. Recovery, survival and functionalevaluation by transplantation of frozen-thawed mouse germ cells. HumanReprod. 2004, 19: 948-53, Pegg D E, Principles of Cryopreservation.Methods Mol Biol. 2007; 368: 39-57, the contents of which areincorporated by reference. Thus, in a further embodiment, the carriermedium further comprises an agent or cryoprotectorant selected from oneor more of dimethyl sulfoxide (DMSO), glycerol, sucrose and othersugars. Alternative cryoprotectants include dimethylacetamide as analternative to glycerol, trehalose and/or sucrose. In a furtherembodiment, the carrier medium comprises conventional cryoprotectants,optionally in combination with growth factors and/or differentiationfactors. Examples of suitable carrier mediums can be found in WO9832333,WO9739104 or WO1997/039104.

In a yet further embodiment, the carrier medium further comprises apropriety preparations such as Profreeze® and CryoStor®. Profreeze issold by Lonza-BioWhittaker as freezing medium containing components ofnon-animal origin. The carrier medium may be supplemented with theagents or protectorants discussed herein, in particular DMSO, glycerol,sucrose and other sugars, and further in particular DMSO. In aparticular embodiment, the carrier medium includes Profreeze® CDNAOFreezing Medium, optionally in combination with DMSO. In a furtherembodiment, the carrier medium comprises Alpha MEM, Profreeze® CDNAOFreezing Medium and DMSO.

The present invention contemplates and includes the possibility that thecarrier medium fulfils multiple requirements. Thus, the carrier mediummay function as both a culture medium and a cryopreservation medium.Equally, the carrier medium may function as both a cryopreservationmedium and a pharmaceutically acceptable carrier.

The carrier medium may comprise dimethylsulfoxide (DMSO) and/orglycerol. In one embodiment, the carrier medium comprisesdimethylsulfoxide (DMSO). In a further embodiment, the compositioncomprises 1-20% DMSO. In a yet further embodiment, the compositioncomprises 1-15% DMSO, 5-15% DMSO, 1-10% DMSO, 5% DMSO, 7.5% DMSO, 10%DMSO, 15% DMSO or 20% DMSO. In a particular embodiment, the DMSO is highpurity grade DMSO.

Some cells may be adversely affected by prolonged contact with DMSO.This can be reduced or eliminated by adding the DMSO to the cellsuspension at 4° C. and removing it immediately upon thawing.Alternatively, a lower concentration of DMSO can be used.

As a further possibility the carrier medium may comprise glycerolinstead of DMSO. Thus, in an alternative embodiment, the carrier mediummay comprise glycerol. In one embodiment, the glycerol is present at aconcentration of 1-30%, 1-20%, 5-20%, 1-15%, 5-15%, 1-10% or 5-10%.

In one embodiment, the medium contains DMSO or glycerol in combinationwith DMEM, HETA-Starch and/or human serum components and/or otherbulking agents.

In one embodiment, the carrier medium is acceptable for injection anddoes not affect the functionality of the cells. In one embodiment, themedium contains serum. In a further embodiment, the medium is a serumfree medium.

In one embodiment, the carrier medium contains serum, in one embodimenthuman serum components. In an embodiment, the carrier medium furthercomprises foetal bovine serum (FBS). In one embodiment, the compositioncomprises 1-50% FBS. In a further embodiment, the composition comprises1-20% FBS, 1-10% FBS, 5% FBS, 30 7.5% FBS, 10% FBS, 15% FBS or 20% FBS.Alternatively, suitable serum includes BSA, transferin and/or egg yolkproteins at the same possible concentrations.

An example of a serum based cryopreservation medium would be a carriermedium comprising an aqueous solution such as Ringer's solution,physiological buffered saline (PBS), Locke's solution, Hank's solution,alpha MEM, DMEM or HAMS F12 together with a cryoprotectorant such asdimethyl sulfoxide (DMSO), glycerol, trehalose, sucrose and other sugarsor dimethylacetamide and serum such as FBS.

Thus, an example serum based cryopreservation medium would be a carriermedium comprising DMEM or alpha MEM, DMSO and serum (using, for example,foetal bovine serum).

The carrier medium may also be serum free and/or protein free and may bea chemically defined media. Examples of serum free media include,KnockOut™ Serum Replacement, KnockOut™ D-MEM, StemPro®-34 SFM.

An example of a serum-free medium would be a carrier medium comprisingan aqueous solution such as Ringer's solution, physiological bufferedsaline (PBS), Locke's solution, Hank's solution, alpha MEM, DMEM or HAMSF12 together with a cryoprotectorant such as dimethyl sulfoxide (DMSO),glycerol, trehalose, sucrose and other sugars or dimethylacetamide and acryopreservation medium such as propriety preparations like Profreeze®or CryoStor®.

Thus, an example serum based cryopreservation medium would be a carriermedium comprising alpha MEM, DMSO and Profreeze® or simply DMSO andProfreeze®.

In one embodiment, the carrier medium comprises Profreeze®. Profreeze®is a serum-free freezing medium and is specifically formulated forcryopreserving cells that have been propagated in serum-free media. Thisprotein-free, non-animal component medium is free of natural animalproteins and maintains high cell viability upon recovery from frozenstorage. In a further embodiment, the carrier medium comprisesProfreeze® together with DMSO, with the DMSO optionally at 7.5 or 15%.Alternatively, the carrier medium may include CryoStor®.

In one embodiment, the medium may contain buffers. Buffers include DMEM,phosphate buffers, or CMF-PBS. Commonly used physiological buffers areall encompassed by the present invention. Example buffers can be foundin the literature, for example, Lelong I H and Rebel G. pH drift of“physiological buffers” and culture media used for cell incubationduring in vitro studies. J Pharmacol Toxicol Methods. 1998; 39: 203-210;John A Bontempo. Development of Biopharmaceutical Parenteral DosageForms. in Drugs and the Pharmaceutical Sciences. Marcel Dekker Inc, NY(ISBN: 0-8247-9981-X): pp 91-108, the contents of which are incorporatedherein by reference.

The medium may optionally further comprise saccharides includingdextran, trehalose, sucrose or dimethylacetamide (DMA).

In one embodiment, the composition comprises progenitor cells;polysulfated polysaccharides and a carrier medium comprising:

-   -   an aqueous medium selected from water, saline, aqueous dextrose,        lactose, Ringer's solution, a buffered solution, hyaluronan,        glycols, physiological buffered saline (PBS), Locke's solution,        Hank's solution, alpha MEM, DMEM, or HAMS F12;    -   a cryopreservation medium, including Profreeze® or CryoStor; and    -   a cryoprotectorant selected from dimethylsulfoxide (DMSO) and/or        glycerol.

In one embodiment, the cryopreservation medium is Profreeze®.

In one embodiment, the aqueous medium is alpha MEM or DMEM.

In one embodiment the cryoprotectorant is DMSO.

In one embodiment, the aqueous medium is present in 1-99%, about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, or about 90%.

In one embodiment, the cryopreservation medium is present in 1-99%,about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about70%, about 80%, or about 90%.

In one embodiment, the cryoprotectorant is present in an amount of1-50%, 1-30%, 1-15%, 1-10%, 1-7.5%, 2.5%, 5%, 7.5%, 10%, 12.5%, 15%,17.5% or 20%.

In a further embodiment, there is provided progenitor cells togetherwith a polysulfated polysaccharide or biologically active molecularfragment thereof, cryopreserved in about 5 mL of Profreeze® CDNAOFreezing Medium, 7.5% DMSO, and 50% Alpha MEM. Cell concentrations atthe time of cryopreservation may be 90×10⁶ cells/cryobags to 180×10⁶cells/cryobag in 5 mL of freezing medium.

In yet another embodiment, the carrier medium comprises a supportmatrix. The support matrix can otherwise be referred to as biomatrix orbioscaffold.

In another embodiment, the present invention provides a method ofenhancing cryopreservation of progenitor cells, comprising exposing theprogenitor cells to a polysulfated polysaccharide or biologically activemolecular fragment thereof.

In another embodiment, the present invention provides the use of apolysulfated polysaccharide or biologically active molecular fragmentthereof to enhance the cryopreservation of progenitor cells.

In another embodiment, the present invention provides a method ofimproving the viability of progenitor cells, comprising exposing apolysulfated polysaccharide or biologically active molecular fragmentthereof to the progenitor cells.

In another embodiment, the present invention provides the use of apolysulfated polysaccharide or biologically active molecular fragmentsthereof to improve the viability of progenitor cells.

In another embodiment, the present invention provides the use of apolysulfated polysaccharide as a cryopreservation agent.

In a further embodiment, the present invention provides the use of acomposition as defined herein to enhance the cryopreservation ofprogenitor cells. In another embodiment, the present invention providesa method of improving the viability of progenitor cells, comprisingcryofreezing a composition as defined herein and subsequently thawingthe composition.

Thus, for the first time, it has been shown that the addition ofpolysulfated polysaccharides to cryogenic media does not have an adverseeffect on the progenitor cells and does not have a detrimental effect ontheir viability. In fact, the addition of polysulfated polysaccharidesto cryogenic media has been shown to enhance viability of the progenitorcells.

Furthermore, it has been shown that the addition of polysulfatedpolysaccharides to progenitor cells maintains or improves the viabilityof the progenitor cells per se. Thus, in a further embodiment there isprovided the use of polysulfated polysaccharides to maintain or improvethe viability of progenitor cells. In a further embodiment, there isprovided a method of maintaining or improving the viability ofprogenitor cells comprising contacting a polysulfated polysaccharide tothe progenitor cell.

It has also been found that polysulfated polysaccharides or biologicallyactive molecular fragments thereof can regulate the proliferation ofprogenitor cells.

Thus, in another embodiment, the present invention provides a method ofregulating the proliferation of progenitor cells, comprising exposing apolysulfated polysaccharide or biologically active molecular fragmentthereof to a progenitor cell.

In another embodiment, the present invention provides the use of apolysulfated polysaccharide or biologically active molecular fragmentthereof to regulate the proliferation of progenitor cells.

In a further embodiment, the present invention provides the use of acomposition as defined herein to increase proliferation. Thus, inanother embodiment, the present invention provides a method ofregulating the proliferation of progenitor cells, comprising using acomposition as defined herein. In another embodiment, the presentinvention provides the use of a composition as defined herein toregulate the proliferation of progenitor cells.

In one embodiment, proliferation is increased or upregulated.

Thus, for the first time, it has been shown that the use of polysulfatedpolysaccharides with progenitor cells can improve the proliferation ofthe progenitor cells. The polysulfated polysaccharides stimulateprogenitor cell proliferation in a concentration dependent manner.Polysulfated polysaccharides can therefore be used in applications whereit is desired to proliferate the cells. For example, in vitroproliferation of progenitor cells would be useful for expansion of thecolony for application in the field of bio-engineering. As an example, abioscaffold could be impregnated with a colony of progenitor cells andperfused by a culture medium at 37° C. containing polysulfatedpolysaccharide(s). This would promote proliferation to further engraftand fill the scaffold thereby providing a more functional substitutetissue. As a further example, a pre-shaped (tubular or hemi-spherical)bioscaffold could be seeded with autologous or allogeneic progenitorcells and perfused with media containing polysulfated polysaccharide(s)to eventually produce a trachea or joint surface for transplantation ina host where these cartilages are defective. Further information onthese uses can be found in Chen F H and Tuan R S. Mesenchymal cells inrhematic diseases. Arthritis research and Therapy. 2008; 10: 223-239.

In vivo polysulfated polysaccharide stimulation of proliferation wouldbe advantageous to facilitate engraftment into large defects (such as injoint cartilage) or compartments denuded of viable endogenous residentcells (eg the centre of the intervertebral disc) thereby reducing thetime required for repair and reconstitution of the defect. Sinceprogenitor cells are also a bountiful source of anti-inflammatorycytokines and immunosuppressive factors (see for example Aggarwal S andPittenger A, Human progenitor cells modulate allogeneic immune cellresponses. Blood. 2005; 105: 1815-1822, Tyndale A, et al.Immunomodulatory properties of progenitor cells: a review based on aninterdisciplinary meeting held at the Kennedy Institute of RheumatologyDivision, London, UK, 31 Oct. 2005. Arthritis Res Ther. 2007; 9: 301-15;Jorgensen C, et al. Multipotent mesenchymal stromal cells in articulardisease. Best Practice and Research Clinical Rheumatology. 2008; 22: 269284) their proliferation at sites of inflammation or antigenic responsefollowing injection would increase the potential for suppression ofthese unwanted cellular processes.

It has also been found that polysulfated polysaccharides can regulatedifferentiation of progenitor cells. The differentiation can beupregulated or downregulated.

In one embodiment there is provided a method of regulatingdifferentiation of progenitor cells by exposing a polysulfatedpolysaccharide or biologically active molecular fragment thereof to aprogenitor cell.

In a further embodiment, there is provided the use of a polysulfatedpolysaccharide or biologically active molecular fragment thereof toregulate the differentiation of progenitor cells.

The present invention regulates differentiation of progenitor cells. Thecells of the present invention can differentiate into chondrocyte,osteoblast, and adipocyte lineages and in one embodiment candifferentiate into cell types of different lineages, including bone,cartilage, adipose, muscle, tendon, and stroma.

In particular, in one embodiment of the present invention, thepolysulfated polysaccharides or biologically active molecular fragmentsthereof can regulate differentiation into chondrocytes. In a furtherembodiment, the polysulfated polysaccharides or biologically activemolecular fragments thereof can regulate differentiation intoosteoblasts. In a yet further embodiment, the polysulfatedpolysaccharides or biologically active molecular fragments thereof canregulate differentiation into adipocytes. In a further embodiment, thepolysulfated polysaccharides or biologically active molecular fragmentsthereof can regulate differentiation into fibrochondrocytes. In afurther embodiment, the polysulfated polysaccharides or biologicallyactive molecular fragments thereof can regulate differentiation intotenocytes. In a further embodiment, the polysulfated polysaccharides orbiologically active molecular fragments thereof can regulatedifferentiation into cardiocytes.

In particular, it has been found that polysulfated polysaccharides orbiologically active molecular fragments thereof can regulate and inducechondrogenesis.

In a further embodiment, the present invention regulates chondrogenesisin progenitor cells which support chondrocyte phenotype differentiationand survival. Therefore, in one aspect, the present invention relates tothe formation of chondrocytes or fibrochondrocytes.

In a further embodiment the polysulfated polysaccharide upregulatesdifferentiation. In an alternative embodiment, the polysulfatedpolysaccharide downregulates differentiation.

In one embodiment of the present invention there is provided a method ofregulating chondgrogenesis in progenitor cells comprising applying apolysulfated polysaccharides to a progenitor cell.

In a further embodiment there is provided the use of a polysulfatedpolysaccharide to regulate chondrogenesis in progenitor cells.

In a further embodiment there is provided the use of a polysulfatedpolysaccharide to down regulate osteogenesis in progenitor cells.

In a further embodiment there is provided the use of a polysulfatedpolysaccharide to prevent osteogenesis by progenitor cells. This methodor use may find application where production of bone would be harmful tothe host such as at soft tissue sites which require flexibility andmovement for normal function eg. muscle (heart), stroma, supportiveconnective tissues etc. or at sites where bone could impinge or entrapnerve fibres/roots or blood vessels leading to parathesis, paralysis,ischemia and potential irreversible tissue injury.

In a further embodiment there is provided a method of treating a cell toundergo chondrogenesis comprising contacting a progenitor cell with aneffective amount of a polysulfated polysaccharide or a biologicallyactive molecular fragment thereof, for a time and under conditions thatstimulate the cell to differentiate.

Progenitor Cells

The term “progenitor cell” is intended to encompass any multipotentcell. Thus, the term progenitor cell encompasses adult and embrionicstem cells.

In one embodiment, the progenitor cell is a mesenchymal progenitor cell.

In a further embodiment, the progenitor cell is an endogenous orexogenous embryonic or adult mesenchymal or mesenchymal progenitor cell.In a further embodiment, progenitor cell is a multipotent stromal cell.In a further embodiment, the cell is an adult undifferentiatedmesenchymal cell.

In a further embodiment, the progenitor cell is a chondroprogenitorcell.

In one embodiment, the progenitor cells are derived from bone marrow.Alternatively, the progenitor cells are derived from cartilage, synovialtissue, muscle, adipose tissue, skin, umbilical cord, dental pulp, orother available sources.

In one embodiment the progenitor cell is a somatic cell, such asconnective tissue cells repressed from differentiation by endogenousfactors.

In a further embodiment, the progenitor cells are a population of cellsenriched for Stro-1^(bri), or homogeonous Stro-1^(bri) cells, orStro-1^(bri) progeny cells.

Polysulfated Polysaccharides

The polysulfated polysaccharide family can be considered to be anynaturally occurring or semi-synthetic/synthetic polysulfatedpolysaccharide or a biologically active fragment thereof that containstwo or more sugar rings or carbohydrate structures to which one or moresulfate ester groups are covalently attached as exemplified by heparinand pentosan polysulfate.

According to one embodiment, the polysulfated polysaccharide orbiologically active fragment thereof can be selected from, but are notlimited to, naturally occurring high molecular weight heparin, lowmolecular weight heparins, heparan sulfate, pentosan polysulfate,chondroitin polysulfate, chitosan polysulfate, dermatan polysulfatesulodexide, dextran polysulfate, polysulfated insulin, sulfatedlactobionic acid amide, sulfated bis-aldonic acid amide, sucroseoctasulfate, fucoidan-1, fucoidan-2, sulfated beta-cyclodextrin,sulfated gamma-cyclodextrin and small sulfated compounds including, butare not limited to, inositol hexasulfate.

In a further embodiment, the polysulfated polysaccharides include:pentosan polysulfate chondroitin polysulfate, chitosan polysulfate,dextran polysulfate and heparin (high and low molecular weight).

In a yet further embodiment, the polysulfated polysaccharides arepentosan polysulfate, dextran polysulfate and heparin.

In a yet further embodiment, the polysulfated polysaccharides arepentosan polysulfate, the sodium salt of pentosan polysulfate (NaPPS),the magnesium salt of pentosan polysulfate (MgPPS), and/or the calciumsalt of pentosan polysulfate (CaPPS).

One particular polysulfated polysaccharide is pentosan polysulfate (PPS)or its sodium salt. Pentosan polysulfate has been shown to improve theviability of progenitor cells, enhance the cryopreservation ofprogenitor cells, regulate the proliferation of progenitor cells and/orregulate the differentiation of progenitor cells. Pentosan polysulfatehas been shown to upregulate differentiation and in particular inducechondrogenesis.

An alternative polysulfated polysaccharide is dextran polysulfate.Dextran polysulfate has been shown to downregulate or repressdifferentiation and in particular downregulate or represschondrogenesis.

Uses

The progenitor cells of the present invention can differentiate into anumber of cell types including chondrocytes, fibrochondrocytes,osteoblasts and adipocytes.

Since progenitor cells can differentiate into chondrocytes orfibrochondrocytes, these cells are useful in the production ofextracellular matrix. Extracellular matrix may be suitable fortransplantation into a connective tissue defect in a subject in need ofsuch a treatment.

The present invention can be used to induce cartilage repair,restoration or matrix neogenesis and attenuate its catabolism byadministering a polysulfated polysaccharide or a biologically activemolecular fragment thereof in combination with progenitor cells.

In a further embodiment, the compositions of the present invention canalso be used as an immunosuppressant, anticatabolic or anti-inflammatoryagent. As an example, the composition of the present invention may beused in the treatment of rheumatoid arthritis.

The methods discussed herein can be used in vivo or in vitro. In vivo,the inserted progenitor cells may, among other things, rebuild cartilagein-situ. In addition, the resident progenitor cells in the joints mayalso be stimulated to rebuild cartilage in-situ thus forming aneffective directed treatment.

In vitro, the present invention allows for the production of cartilagewithin a biomatrix that can subsequently be implanted into a patient.This could be used to generate cartilage to partially or totally replacearticulating joint surfaces, for the replacement ofcartilaginous/fibrocartilagenous tissues or any other tissues that mightbenefit from this process which have been injured or arise from geneticabnormalities that require surgical correction.

The present invention also finds use with patients that may not benefitfrom the medical or surgical treatments currently available. Forexample, many sportspersons or individuals who have suffered from acuteinjury caused by trauma may have cartilage/fibrocartilagenous defectswhich are symptomatic. The present invention provides a method thatcould be used to stimulate growth of new cartilage to replace thedefective tissue. This can either be done in vivo by stimulatingprogenitor cell growth in the joint in-situ or in vitro via a suitablebioscaffold which is shaped so as to fit into the defect andsubsequently inserted into the defect; or by both in vivo and in vitromethods. Alternatively it could be used with older patients withestablished joint degeneration such as in osteoarthritis of theperipheral joints and spine where the present invention could be used tostimulate growth of new cartilage to replace the defective tissue andprevent progression of osteophytes and reduce the inflammation which isoften the cause of symptoms of these disorders.

In one embodiment, the present invention has identified compound(s) thatcan act as both a cryopreservation agent and as an agent which canregulate differentiation. In a further embodiment, the present inventionhas identified compound(s) that can act as both a cryopreservation agentand as an agent which can regulate proliferation. In a furtherembodiment, the present invention has identified compound(s) that whichcan regulate proliferation and regulate differentiation. In a furtherembodiment, the present invention has identified compound(s) that canact as a cryopreservation agent, as an agent which can regulateproliferation and as an agent which can regulate proliferation. Suchmulti-use compounds have not been previously found in relation toprogenitor cells.

The present invention has identified families of molecules or theirbiologically active fragments that can independently or in combinationwith each other enhance the cryopreservation of progenitor cells,regulate their cell proliferation and or regulate their differentiation;thus, these molecules can be used in combination with progenitor cellsin therapeutic treatment.

In particular, the present invention allows the progenitor cells todifferentiate into chondrocytes/fibroblasts thereby allowing for theformation of cartilage or fibrocartilage. Therefore, the use ofprogenitor cells and polysulfated polysaccharide can be used to treatdegenerative diseases, to treat cartilage/fibrocartilage defect and/orto preventing or minimising the progression of degenerative diseases andcartilage defects.

The present invention has identified a novel composition. Thiscomposition has therapeutic use and can be advantageously used either invivo or in vitro. In one embodiment, the method is carried out in vivo.Alternatively, the method is carried out in vitro.

Therefore, in one embodiment of the present invention there is provideda composition as described herein for use as a medicament.

In a further embodiment of the present invention there is provided acomposition as described herein for use in the treatment of any diseasethat is affected by a breakdown or reduction of cartilage, includingdiseases of the musculoskeletal system including rheumatoid arthritis(RA), osteoarthritis (OA), and intervertebral disc degeneration (DD),degenerative diseases, and method of inducing cartilage repair,restoration or matrix neogenesis.

In a further embodiment of the present invention there is provided acomposition as described herein for use in the treatment of any diseasethat is affected by a breakdown or reduction of cartilage, includingdiseases of the musculoskeletal system including rheumatoid arthritis(RA), osteoarthritis (OA), and intervertebral disc degeneration (DD),degenerative diseases, and method of inducing cartilage repair,restoration or matrix neogenesis.

In a further embodiment of the present invention there is provided acomposition as described herein for use in the treatment of any diseasewhere the differentiation of progenitor cells via osteogenesis isunwanted. For example, bone formation is often unwanted for soft tissuerepair such as intra-discal injection. Leakage of the cells from a discinto the spinal canal or onto the adjacent organs (eg oesophagus) can bedisastrous. One example of this was seen when BMP-2 was placed in thedisc space to promote spinal fusion (new bone). It was found that theuse of recombinant bone morphogenetic proteins resulted in lifethreatening complications, due to ectopic bone formation adjacent to thedisc space which caused airway and neurological compression.

In a further embodiment of the present invention there is provided acomposition as described herein for use in the treatment of a diseasethat is affected by a breakdown or reduction of adipose tissue.

In a further embodiment of the present invention there is provided theuse of a composition as described herein for the manufacture of amedicament for the treatment of any disease that is affected by abreakdown or reduction of cartilage, including diseases of themusculoskeletal system including rheumatoid arthritis (RA),osteoarthritis (OA), and intervertebral disc degeneration (DD),degenerative diseases, and method of inducing cartilage repair,restoration or matrix neogenesis.

Therefore, according to one embodiment of the present invention there isprovided a method of treating, mitigating, reducing or preventing anydisease that is affected by a breakdown or reduction of cartilage suchas diseases of the musculoskeletal system including rheumatoid arthritis(RA), osteoarthritis (OA), and intervertebral disc degeneration (DD),degenerative diseases, and method of inducing cartilage repair,restoration or matrix neogenesis, comprising administering atherapeutically effective amount of a composition as defined herein.

Examples of such an application would be to inject the composition ofthe present invention into joints of individuals with cartilage or disclesions or systemically for other less accessible sites, allowing thepreparation to perfuse the tissue and cells thereby exerting its uniquebiological effects. Applications could include treating individuals whomay not have clinical defined disease (often OA or related disorders)but have sustained a traumatic injury to joint tissues through, forexample, sport or work-related activity.

The methods and uses of the present invention could also serve as aprophylactic method following arthroscopic or open surgery wherecartilage or meniscal excision/debridement was necessary. It is wellestablished that with time such post surgical patients will generallyprogress to exhibit symptomatic OA requiring medical treatment. It isnot unlikely that by diminishing cartilage degradation symptoms may alsoimproved because of the reduction in production of antigens whichpromote inflammation.

Thus, use of the compositions of the present invention discussed hereinto regulate differentiation and/or cell proliferation of progenitorcells introduced into the patient can be used to treat, mitigate, reduceor prevent any disease that is affected by a breakdown or reduction ofcartilage. Specific diseases include diseases of the musculoskeletalsystem including rheumatoid arthritis (RA), osteoarthritis (OA), andintervertebral disc degeneration (DD), degenerative diseases, and methodof inducing cartilage repair, restoration or matrix neogenesis.

In one embodiment the composition is administered intravenously.According to a yet further embodiment, the composition is administeredsystemically. According to a yet further embodiment, the composition isadministered intra-articularly. According to a yet further embodiment,the composition is administered intra-discally. According to a yetfurther embodiment, the composition is administered systemically.

In one embodiment is the method of injecting a polysulfatedpolysaccharide, in combination with progenitor cells, into the joint(s)of the patient. The polysulfated polysaccharide helps regulatedifferentiation and/or proliferation of the progenitor cells.

It has additionally been found that polysulfated polysaccharides of thepresent invention can also produce, upregulate or stimulate theproduction of hyaluronan or hyaluronic acid (HA) in the differentiatedcells. The hyaluronic acid (HA) can be produced in an animal or cell,namely an animal in vivo or in a cell in vitro.

This unexpected finding means that the compositions of the presentinvention can be used to replace HA lost in joints, particularlysynovial fluid, ether due to normal wear and tear, degenerative diseasesor other acute traumas. As synovial fluid degenerates, its ability toprotect and lubricate joints is reduced. This degrades the joint furtherand can also stimulate the production of autoantigens which causes yetfurther damage. In the past, one way of overcoming or at leastmitigating this problem has been to replace the synovial fluid.

However, the present invention provides a means of stimulating theproduction of hyaluronan or hyaluronic acid (HA) without the need toreplace the synovial fluid itself. The compositions of the presentinvention can be contacted with progenitor cells, which thendifferentiate into mesenchymal cells (e.g. chondrocytes or fibroblasts)to increase the production of HA. The HA is formed in situ and can beused to replace HA lost in synovial fluid which treats, reduces or atleast mitigates the damage caused by the degenerative diseases or tissuedegradation.

Thus, according to a further embodiment, the present invention relatesto a method of producing, upregulating or stimulating the production ofhyaluronan (HA), comprising administering a composition as definedherein.

In one embodiment of the present invention, the compositions methods anduses can be used to treat arthritis or other degenerative diseases.However, in an alternative embodiment, the present invention excludesmethods to treat arthritis or other degenerative diseases. Specifically,in one aspect, the present invention includes the use of a polysulfatedpolysaccharide or biologically active molecular fragment thereof incombination with progenitor cells to treat arthritis or otherdegenerative diseases.

Thus, in one embodiment of the present invention, there is provided amethod of treating a patient suffering from diseases of themusculoskeletal system including rheumatoid arthritis (RA),osteoarthritis (OA), and intervertebral disc degeneration (DD);degenerative diseases, osteoarthritis of synovial joints, ophthalmology,prevention of post-surgical abdominal adherences, skin treatment andrepair and restoration of the function of the extracellular matrix; orfor inducing cartilage repair, restoration or matrix neogenesis;comprising administering a composition as defined herein, to regulatedifferentiation and/or proliferation of the progenitor cells.

The patient or subject can be a human or animal patient. In oneembodiment, the patient is a mammal including a human, horse, dog, cat,sheep, cow, or primate. In one embodiment the patient is a human. Thepatient may suffer from a degenerative disease and/or a cartilagedefect. The patient may be an athlete or may have been subjected to atrauma causing joint damage.

The present invention encompasses methods of treatment involvingpolysulfated polysaccharides and progenitor cells. The present inventionalso encompasses the polysulfated polysaccharides and progenitor cellsfor use as a medicament; the use of the polysulfated polysaccharides andprogenitor cells in the manufacture of a medicament for the treatmentsas discussed herein; and also compositions and formulations containingthe polysulfated polysaccharides and progenitor cells.

Combination Therapies

The present invention has also identified for the first timepolypeptides or a biologically active fragment thereof which can alsoregulate differentiation and cell proliferation, particularly regulatechondrogenesis and cell proliferation. The polypeptide is anon-collagenous NC4 domain of alpha IX collagen or a biologically activemolecular fragment thereof (hereinafter NC4). The term “a biologicallyactive fragment” is synonomous with the term “a biologically activemolecular fragment”.

Surprisingly, this invention has discovered that while the two separatefamilies can exert their regulation of chondrogenesis and cellproliferation independently when combined together they can actsynergistically, not only to increase their individual effects but toafford greater specificity of action.

These unexpected findings opens up the possibility of using apolysulfated polysaccharide in combination with NC4, in a number of newapplications since by regulating chondrogenesis, it is possible, amongother things, to rebuild cartilage and intervertebral discs, prevent thedegradation of joints and enhance the repair of avascular connectivetissues. Prior to the present invention, it was not known that acombination of a polysulfated polysaccharides and NC4 could regulatechondrogenesis and cell proliferation, and it was certainly not knownthat the combination would have a synergistic effect.

Accordingly, in a further embodiment, the present invention provides acomposition comprising progenitor cells together with a polysulfatedpolysaccharide or biologically active molecular fragment thereof and NC4or a biologically active molecular fragment thereof.

In a further embodiment, the composition further comprises a carriermedium, culture medium, cryopreservation medium and/or pharmaceuticallyacceptable carrier.

Thus, in a further embodiment, the present invention provides acomposition comprising progenitor cells, a polysulfated polysaccharideor biologically active molecular fragment thereof and NC4 or abiologically active molecular fragment thereof, together with a carriermedium.

The carrier medium may be a culture medium, cryopreservation medium orpharmaceutically acceptable carrier.

Any reference to the compositions of the present invention which relateto progenitor cells and polysulfated polysaccharides also relate tocomposition containing progenitor cells, polysulfated polysaccharidesand NC4, including the concentrations, cell numbers and/or types andamounts of optional further ingredients. In addition, the methods anduses of the compositions as defined herein also relate to thecomposition including both a polysulfated polysaccharide and NC4.

Thus, according to a further embodiment of the present invention, thereis provided a method of regulating chondrogenesis and/or cellproliferation comprising administering a composition comprisingprogenitor cells, a polysulfated polysaccharide or biologically activemolecular fragment thereof and NC4 or a biologically active molecularfragment thereof.

Thus, in another embodiment, the present invention provides a method ofregulating the proliferation of progenitor cells, comprising exposing apolysulfated polysaccharide or biologically active molecular fragmentthereof and NC4 or a biologically active molecular fragment thereof to aprogenitor cell.

Thus, in another embodiment, the present invention provides a method ofregulating differentiation of progenitor cells by exposing apolysulfated polysaccharide or biologically active molecular fragmentthereof and NC4 or a biologically active molecular fragment thereof tothe progenitor cells.

In one embodiment, the biologically active molecular fragment of NC4 hasat least 65% amino acid identity to a fragment of SEQ ID NO:1.

In a further embodiment, the biologically active molecular fragment ofNC4 has at least 65% amino acid identity to a fragment of SEQ ID NO:2.

In a yet further embodiment, the biologically active molecular fragmentof NC4 has at least 65% amino acid identity to a fragment of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO 8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ IDNO:24.

In a further embodiment, biologically active molecular fragment of NC4has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100% aminoacid identity to the frangments listed above.

It is also possible to administer or use the compositions of the presentinvention as part of a combination therapy. For example thepolysaccharide(s) of the present invention may be administered incombination with one or more other compounds. These compounds may be astructure modifying osteoarthritis drug (SMOADs).

The present invention extends to combination therapies for use in thetreatment of the diseases discussed herein. Particularly, in oneexample, the present invention extends to the use of a polysulfatedpolysaccharide in combination with a further agent for use in thetreatment of various degenerative conditions. It should be understoodthat these agents can be administered at the same time or a differenttime. Thus the combination therapy may comprise the active agents beingadministered at the same time either in a single formulation or inmultiple formulations administered at the same or different times.Equally, the combination therapy may comprise the active agents beingadministered in different formulations at different times. Theformulations could be administered sequentially and may be separated bya period of time including hours, days, weeks and months.

The present invention also extends to the use of the polysulfatedpolysaccharides as discussed herein in combination with one or moregrowth factors. The present invention further extends to the methods,uses, formulations and/or compositions as disclosed herein incombination with one or more growth factors. Possible growth factorsinclude insulin like growth factor, insulin, fibroblast like growthfactors; BMP-TGF-beta super family (eg, BMP-2, BMP-7, BMP-8, TGF beta)and fibroblast growth factor family, IGF, FGF, EGF, PDGF and VEGF.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The Effects of Pentosan Polysulfate (PPS) on Normal Human MSCsFreeze/Thaw Viabilities. MSCs were rapidly thawed in a 37° C. water bathand washed twice with HHF (HBSS containing 5% (v/v) Foetal Calf Serum).The cells were subsequently seeded into multiple T-75 flasks at 8,000cells/cm². The cells were grown until 70-80% confluent, trypsinised andcryopreserved at cell concentrations of 50×10⁶/ml in Profreeze®/7.5%DMSO supplemented with PPS at the indicated concentrations. Ampouleswere retrieved from liquid nitrogen storage and rapidly thawed, gentlymixed and 10 ul samples removed at time=0, 30, 60, 90 and 120 minutes.To each cell sample, 290 ul of trypan blue was added and cellcounts/viability testing performed. PPS did not adversely affect cellviability.

FIG. 2. Bar graph showing the viability of different numbers of murineATDC5 progenitor cells suspended in cryogenic media containing 7.5% DMSOand various concentrations of Pentosan Polysulfate (PPS) after beingsubjected to freeze-thawing cycle. Cell viability was determined usingthe mitochondrial dehydrogenase MTT assay.

FIG. 3. Effects of different concentrations of Pentosan Polysulfate(PPS) on progenitor cell viability following cryopreservation in liquidnitrogen and rapidly thawing as described in FIG. 1. Cell viability wasdetermined using the mitochondrial dehydrogenase MTT assay. Datashown=Means±SD *=p<0.05 relative to control values

FIG. 4. The Effects of Pentosan Polysulfate (PPS) on Human Progenitorcell Proliferation. Primary human progenitor cells were cultured in 24well plates in growth media supplemented with PPS at the indicatedconcentrations. At various time intervals (day 1,3,6), the growth mediawas removed and replaced with phenol red free media containing thetetrazolium salt WST-1 for 2 hours at 37° C/5% CO2. WST-1 is cleaved bymitochondrial dehydrogenase in viable cells to produce a formazan dyethat can be detected using an ELISA plate reader at a wavelength of 450nm. Absorbance at 450 nm for each time point is shown for allconcentrations of PPS. A statistically significant increase inproliferation was observed on day 6 at concentrations of PPS in excessof 1 μg/ml (*p<0.01, ANOVA).

FIG. 5. A bar graph of the concentration dependent effects of PentosanPolysulfate (PPS) on DNA synthesis by human progenitor cells, asdetermined by the incorporation of ³H-Thymidine into macromolecular DNA,after 4 day micromass cultures. *=p<0.05; **=p<0.005 relative tocontrols.

FIG. 6. The Effects of Pentosan Polysulfate (PPS) on Human progenitorcells treated with apoptotic agents. Human progenitor cells were platedin serum-free media supplemented with PPS at the indicatedconcentrations. Progenitor cell apoptosis was induced by the addition ofa combination of 30 ng/ml IL-4 plus 30,000 U/ml IFN-gamma. Following 5days culture, cells were harvested by trypsinisation and viabilitiesassessed by Annexin V staining. A two-fold reduction inIFN-gamma/IL-4-induced apoptosis (Annexin V positive cells) is observedwhen progenitor cells are cultured at concentrations of PPS in excess 1ug/ml.

FIG. 7. The Effects of Pentosan Polysulfate (PPS) on Human progenitorcell Differentiation: Mineralisation Assay. Primary human progenitorcells were cultured in 96 wells plates in non-osteoinductive growthmedia (media control) or in osteoinductive conditions (alphaMEMsupplemented with 10% FCS, 100 microM L-ascorbate-2-phosphate,dexamethasone 10⁻⁷ M and 3 mM inorganic phosphate) in the presence ofPPS at the indicated concentrations. On day 28, the concentration ofacid solubilisd calcium per well was determined using theCresolphthalein Complexone method. (A) The concentration of acidsolubilised calcium per microg of DNA/well was determined following theassessment of the total amount of DNA per well using a fluorogenic DNAstain (Hoeshst 33258). A statistically significant decrease inmineralised matrix formation was observed when concentrations of PPS of1 ug/ml and 100 ug/ml were used (*p<0.01, ANOVA). (B) Phase-contrastphotomicrographs of mineralised cultures at ×20 magnification.

FIG. 8. The Effects of Pentosan Polysulfate (PPS) on Human progenitorcell Differentiation: Adipocyte Formation. Primary progenitor cells werecultured in 96 well plates in non-adipogenic growth media (mediacontrol) or under adipogenic conditions (0.5 mMmethylisobutylmethylxanthine, 0.5 μM hydrocortisone, and 60 μMindomethacin) in the presence of PPS at the indicated concentrations. Onday 28, the presence of lipid laden adipocytes was determined using thelipophilic dye Oil Red O. The relative amount of solubilised lipid perμg of DNA/well was determined following the assessment of the totalamount of DNA per well using a fluorogenic DNA stain (Hoeshst 33258).(A) A statistically significant increase in adipocyte number wasobserved at concentrations of PPS in excess lug/ml (*p<0.01, ANOVA). (A)Phase-contrast photomicrographs of Oil Red O labeled adipocytes at ×20magnification.

FIG. 9. Concentration dependent effects of Pentosan Polysulfate (PPS) onMurine Progenitor Cell (progenitor cells C3H10T1/2) biosynthesis ofProteoglycans (PGs) and DNA content when grown in monolayer cultures.Data shown=Means±SD.

FIG. 10. A bar graph of the concentration dependent effects of PentosanPolysulfate (PPS) on DNA synthesis by murine Progenitor Cells (C3H10T1/2cells) grown in monolayer cultures for 2 days as determined by theincorporation of ³H-Thymidine into macromolecular DNA.

FIG. 11. A bar graph of the concentration dependent effects of PentosanPolysulfate (PPS), on the biosynthesis of Proteoglycans (PGs) asdetermined by the incorporation of radioactively labelled sulfate intothe sulfated glycosaminoglycans (³⁵S-GAG) of PGs after 2 day monolayercultures of human progenitor cells. The data was expressed as ³⁵S-GAGradioactivity as decays per minute (DPM) normalised to DNA content.*=p<0.05; **=p<0.005; ***=p<0.0005.

FIG. 12. A bar graph of the concentration dependent effects of PentosanPolysulfate (PPS), on the biosynthesis of Proteoglycans (PGs) asdetermined by the incorporation of radioactively labelled sulfate intothe sulfated glycosaminoglycans (³⁵S-GAG) of PGs in 6 day pelletcultures of murine progenitor cells (ADTC-5). *=p<0.05 relative tocontrol.

FIG. 13. Gene Expression by ATDC5 cells of Type II collagen and Sox-9 in6-day pellet culture incubated with various concentrations of PPS inMaintenance Medium (MM).

FIG. 14. A bar graph of the concentration dependent effects of Heparinon the biosynthesis of Proteoglycans (PGs as determined by theincorporation of radioactively labelled sulfate into the sulfatedglycosaminoglycans (³⁵S-GAG) of PGs in 6 day pellet cultures of murineprogenitor cells (ADTC-5). Heparin does not exhibit a chondrogeniceffect over the concentration range 1.25-20 ug/mL in this cell line.

FIG. 15. Bar graphs showing the concentration dependent effects ofPentosan Polysulfate (PPS), on the biosynthesis of Proteoglycans (PGs)as determined by the incorporation of radioactively labelled sulfateinto the sulfated glycosaminoglycans (³⁵S-GAG) of PGs in 7 day pelletcultures of a murine progenitor cells (C3H10T1-2).

FIG. 16. A bar graph showing the concentration dependent effects of PPSon proteoglycan synthesis by murine progenitor cells (C3H10T1-2) inmicromass cultures for 6 days and 9 days. PPS was included in the media(Ham's F12+10% FCS) and was changed every 48 hours. ³⁵S—SO₄ was added 24hours before culture termination. Synthesis normalized to DNA content.*P<0.05, **P<0.005, ***P<0.0005 relative to controls.

FIG. 17. Bar graphs showing the concentration dependent effects of PPSon proteoglycan synthesis by human progenitor cells in micromasscultures for 5 days. Data is presented as 35S-GAG radioactivity and as apercentage of control taken as 100% . *P<0.05 relative to control.

FIG. 18. A: Bar graph showing the Pentosan Polysulfate (PPS)concentration dependent stimulation of type II collagen production byhuman progenitor cells in micromass cultures for 10 days as determinedby scanning and digital analysis of the immuno stained micromasscultures shown in B. (see text for details).

FIG. 19. Bar graphs showing the concentration dependent effects of (A)Hyaluronan (Supartz™) and (B) Dextran Polysulfate on proteoglycansynthesis by human progenitor cells in micromass cultures for 5 days.Data is presented as ³⁵S-GAG radioactivity and as a percentage ofcontrol, taken as 100% or as DPM/ug DNA. *P<0.05 relative to control.

FIG. 20. Shows the results of culturing primary human progenitor cellgrowth media supplemented with PPS and/or Hyaluronic acid (Supartz™), atthe indicated concentrations. At various time intervals (day 3 & 5), thegrowth media was removed and replaced with phenol red free mediacontaining the tetrazolium salt WST-1 for 2 hours at 37° C./5% CO₂.Absorbance at 450 nm for each time point is shown for all concentrationsof PPS and HA. This experiment shows that HA and PPS do not actsynergistically to stimulate progenitor proliferation.

FIG. 21. A bar graph of the concentration dependent effects of rhNC4(batch PBA-1202P) expressed by K. lactis, in the absence (maintenancemedia, MM) and presence of insulin (10 micrograms/mL) (differentiationmedia, DM), on the biosynthesis of Proteoglycans (PGs) as determined bythe incorporation of radioactively labelled sulfate into the sulfatedglycosaminoglycans (³⁵S-GAG) of PGs after 3 day culture with MurineATDC5 progenitor cells. The data was expressed as % change relative tocontrol cultures that contained no rhNC4. P<0.05 was statisticallysignificant relative to control cultures.

FIG. 22. A bar graph of the concentration dependent effects of rhNC4(batch PBA-1202P) expressed by K. lactis, in the absence (maintenancemedia, MM) and presence of insulin (10 micrograms/mL) (differentiationmedia, DM), on the biosynthesis of Proteoglycans PGs) in pellet culturesof ATDC5 cells. Data is shown as the % change relative to controls takento be 100%.

FIG. 23. A bar graph of the concentration dependent effects of rhNC4(batch PBA-1202P) expressed by K. lactis plus Pentosan Polysulfate (PPS)(2 micrograms/mL), in the absence (maintenance media, MM) and presenceof insulin (10 micrograms/mL) (differentiation media, DM), on thebiosynthesis of macromolecular DNA as determined by the incorporation ofradioactively labelled ³H-Thymidine after 1 day culture with MurineATDC5 progenitor cells. The data was expressed as % change relative tocontrol cultures that contained no rhNC4. P <0.05 was statisticallysignificant relative to control cultures.

FIG. 24. A bar graph of the concentration dependent effects ofcombinations of rhNC4 (batch PBA-1202P) and Pentosan Polysulfate (PPS)on the biosynthesis of ³⁵S-PGs by monolayer cultures of Murine ATDC5progenitor cells. Data is expressed relative to control (maintenancemedia, MM) which was taken as 100%.

FIG. 25. RT-PCR detection of gene expression of the bone marker Runx2,MGP, HAS3, CD44 and the housekeeping gene NADPH expressed by MurineATDC5 progenitor cells cultured in the presence and absence of rhNC4(Batch PBA-1209P) and PPS for 2 days in monolayer cultures.

FIG. 26. RT-PCR detection of gene expression of Runx2 and thetransduction proteins, Smad 2 and Smad 4 and the housekeeping gene NADPHexpressed by Murine ATDC5 progenitor cells cultured in the presence andabsence of rhNC4 (Batch PBA-1209P) and PPS for 2 days in monolayercultures.

FIG. 27. Chromatographic elution profiles showing the effects ofpolysulfated 5 polysaccharides on the biosynthesis of Hyaluronan (HA)and by progenitor cells by measuring the incorporation of ³H-glucosamineinto HA. Superdex-S200 chromatographic profiles of media from humanprogenitor cells cultured in micromass in the presence of variousconcentrations of Pentosan Polysulfate (PPS) for 9 days are displayed inpanels A-D. Before Hyalase digestion the profiles of radioactivity showthe incorporation of ³H-Glucosamine into both HA and PGs by the cellsbut after digestion only the ³H-PGs remain in the void volume of thecolumn. The % difference in areas under the profiles of the digested andpre-digested samples in the void volume fractions represents the amountsof ³H-HA released into the media by the PPS concentration specified.

FIG. 28. A bar graph of the concentration dependent effects of rhNC4(Batch PBA-1202P) expressed by K. lactis yeast cells in the absence andpresence of insulin (10 micrograms/mL) on DNA synthesis as determined bythe incorporation of ³H-Thymidine into macromolecular DNA, after 3 dayculture with Murine ATDC5 progenitor cells. The data is expressed as %change relative to control cultures that contained no rhNC4. P<0.01 wasstatistically significant relative to control cultures.

FIG. 29 A bar graph of the concentration dependent effects of rhNC4(Batch PBA-1202P) expressed by K. lactis yeast cells on Murine ATDC5progenitor cell numbers, determined using a hemocytometer, after 3 dayculture in the absence and presence of insulin (10 micrograms/mL). Thedata is expressed as % change relative to control cultures thatcontained no rhNC4. P<0.01 was statistically significant relative tocontrol cultures.

FIG. 30. A bar graph of the kinetics of ATDC5 cell growth induced byrhNC4 (batch PBA-1200P) (5 ug/ml) or Insulin (10 ug/ml) relative tocontrol over 13 days. 20,000 cells per well were seeded at Day 0 (Start)with medium changes every 48 hrs. Insulin treated cultures reachedconfluence on day 6 and PBA-1200P on day 9. Control cultures alsoreached confluence on day 9 but in contrast to cultures containinginsulin or PBA-1200P ceased to undergo replication.

FIG. 31. A bar graph of the concentration dependent effects of rhNC4(batch PBA-1202P) expressed by K. lactis, in the absence (maintenancemedia, MM) and presence of insulin (10 micrograms/mL) (differentiationmedia, DM), on the biosynthesis of Proteoglycans (PGs) as determined bythe incorporation of radioactively labelled sulfate into the sulfatedglycosaminoglycans (³⁵S-GAG) of PGs after 3 day culture with

Murine ATDC5 progenitor cells. The data was expressed as % changerelative to control cultures that contained no rhNC4. P<0.05 wasstatistically significant relative to control cultures. FIG. 31 showsthat rhNC4 stimulated PG synthesis by the progenitor ATDC5 cells in thepresence and absence of insulin, but more effectively at the higherconcentrations in the absence of insulin.

FIG. 32. A bar graph of the concentration dependent effects of PentosanPolysulfate (PPS), in the presence of insulin (10 micrograms/mL), on thebiosynthesis of Proteoglycans PGs) as determined by the incorporation ofradioactively labelled sulfate into the sulfated glycosaminoglycans(³⁵S-GAG) of PGs after 3 day culture with Murine ATDC5 progenitor cells.The data was expressed as ³⁵S-GAG radioactivity as counts per minute(CPM) and decays per minute (DPM) relative to control cultures thatcontained no PPS. P<0.05 was statistically significant relative tocontrol cultures. FIG. 32 shows the concentration dependent stimulationof PG synthesis by Murine ATDC5 progenitor cells in the presence of PPS.

FIG. 33. A bar graph of the concentration dependent effects of rhNC4(batch PBA-1202P) expressed by K. lactis plus sodium pentosanpolysulfate (PPS) (2 micrograms/mL), in the absence (maintenance media,MM) and presence of insulin (10 micrograms/mL) (differentiation media,DM), on the biosynthesis of Proteoglycans

(PGs) as determined by the incorporation of radioactively labelledglucosamine into the glycosaminoglycans (³H-GAG) of PGs after 1 dayculture with Murine ATDC5 progenitor cells. The data was expressed as %change relative to control cultures that contained no rhNC4. P<0.05 wasstatistically significant relative to control cultures.

FIG. 34. A bar graph of the concentration dependent effects of PentosanPolysulfate (PPS) in the presence of insulin (10 micrograms/mL) on DNAsynthesis (cell replication), as determined by the incorporation of³H-Thymidine into macromolecular DNA after 3 day culture with MurineATDC5 progenitor cells. The data is expressed as 35% change relative tocontrol cultures that contained no PPS.

FIG. 35. SEQ ID NO:1—Amino acid sequence for full length human NC4without signal peptide.

FIG. 36. SEQ ID NO:2—Amino acid sequence for truncated hNC4 obtainedduring 5 expression from K. lactis cultures by action of putative byproline endopeptidase.

FIG. 37. A sequence composition between bovine human, murine and chickNC4 sequences.

DETAILED DESCRIPTION OF THE ILLUSTRATED AND EXEMPLIFIED EMBODIMENTS OFTHE INVENTION

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a polypeptide” includes a plurality ofpolypeptides, including mixtures thereof.

As used herein the term “derived from” shall be taken to indicate that aspecified integer are obtained from a particular source albeit notnecessarily directly from that source.

A “composition” is intended to mean a combination of active agent andanother compound or composition, inert (for example, a detectable agentor label) or active, such as an adjuvant.

Unless the context requires otherwise or specifically stated to thecontrary, integers, steps, or elements of the invention recited hereinas singular integers, steps or elements clearly encompass both singularand plural forms of the recited integers, steps or elements.

The embodiments of the invention described herein with respect to anysingle embodiment shall be taken to apply mutatis mutandis to any otherembodiment of the invention described herein.

Throughout this specification, unless the context requires otherwise,the word. “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated step or element orinteger or group of steps or elements or integers but not the exclusionof any other step or element or integer or group of elements orintegers.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specificexamples described herein. Functionally equivalent products,compositions and methods are clearly within the scope of the invention,as described herein.

The present invention is performed without undue experimentation using,unless otherwise indicated, conventional techniques of molecularbiology, microbiology, virology, recombining DNA technology, peptidesynthesis in solution, solid phase peptide synthesis, and immunology.Such procedures are described, for example, in the following texts thatare incorporated herein by reference:

-   1. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory    Manual, Cold Spring Harbor Laboratories, New York, Second Edition    (1989), whole of Vols I, II, and III;-   2. DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover,    ed., 1985), IRL Press, Oxford, whole of text;-   3. Oligonucleotide Synthesis: A Practical Approach (M. J. Gait,    ed., 1984) IRL Press, Oxford, whole of text, and particularly the    papers therein by Gait, pp 1-22; Atkinson et al., pp 35-81; Sproat    et al., pp 83-115; and Wu et al., pp 135151;-   4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames    & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text;-   5. Perbal, B., A Practical Guide to Molecular Cloning (1984);-   6. Wiinsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls    Metoden der Organischen Chemie (Miiler, E., ed.), vol. 15, 4th edn.,    Parts 1 and 2, 30 Thieme, Stuttgart.-   7. Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir    and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications)

Progenitor Cells

The present invention relates to progenitor cells. In its broadestembodiment, the term progenitor cells is intended to encompass anymultipotent cell. Thus, the term progenitor cell encompasses adult andembrionic stem cell.

The progenitor cell may be a mesenchymal progenitor cell.

The progenitor cell may be an endogenous or exogenous embryonic or adultmesenchymal or mesenchymal progenitor cell. The progenitor cell may be amultipotent stromal cell. The progenitor cell may be an adultundifferentiated mesenchymal cell.

The progenitor cell may be a chondroprogenitor cell.

The progenitor cells may be derived from bone marrow. Alternatively, theprogenitor cells may be derived from cartilage, synovial tissue, muscle,adipose tissue, skin, umbilical cord, dental pulp, or other availablesources.

The progenitor cells may be a somatic cell, such as connective tissuecells repressed from differentiation by endogenous factors.

In a further embodiment, the progenitor cells are a population of cellsenriched for Stro-1^(bri), or homogeonous Stro-1^(bri) cells, orStro-1^(bri) progeny cells.

One type of progenitor cell is a mesenchymal progenitor cell (MPC).Originally derived from bone marrow, MPCs and MPC-like cells have beenidentified to exist in and can be isolated from a large number of adulttissues, where they are postulated to carry out the function ofreplacing and regenerating local cells that are lost to normal tissueturnover, injury, or aging. These tissues include adipose, periosteum,synovial membrane, synovial fluid (SF), muscle, dermis, deciduous teeth,pericytes, trabecular bone, infrapatellar fat pad, and articularcartilage.

MPCs may be defined retrospectively by a constellation ofcharacteristics in vitro, including a combination of phenotypic markersand multipotential differentiation functional properties.

Although plastic adherence serves as the most commonly used and simpleisolation procedure for mesenchymal cells, various positive and negativesurface markers (for example, Stro-1, CD146/melanoma cell adhesionmolecule, CD271/low-affinity nerve growth factor, and stage-specificembryonic antigen-4) have also been used to enrich MPC yield andhomogeneity. In addition, a further panel of surface markers, includingCD140b (platelet-derived growth factor receptor-D), CD340 (HER-2/erbB2),and CD349 (frizzled-9) in conjunction with CD217 can also be used forMPC enrichment.

Further progenitor cells include murine progenitor cells, including celllines C3H10T1/2 and ATDC5 or M111 progenitor cells. C3H10T1/2 areprogenitor stem cell line derived from bone marrow of female C3H/Hemouse strain with fibroblast-like morphology. ATDC5 cells are mouseembryonic derived chondroprogenitor cell line with epithelial-likemorpholology.

The C3H10T1/2 cell line was established in 1973 from 14- to 17-day-oldC3H mouse embryos. These cells display fibroblastic morphology in cellculture and are functionally similar to mesenchymal stem cells.Inhibiting methylation in C3H10T1/2 cells with 5-azacytidine producesstable morphological and biochemical features of muscle, adipose, bone,or cartilage cells It is suggested that this phenotypic alterationresults from activation of endogenous genes in response to blockingmethylation. In addition it has been shown that bone morphogenic protein4 (BMP4), a member of the transforming growth factor type-betasuperfamily, can induce commitment of C3H10T1/2 cells to preadipocytesthat, when subjected to an adipocyte differentiation protocol, developinto cells of the adipocyte phenotype (Tang Qi-Qun, Otto T C, Lane M D.Commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage.Pro Natl Acad Sci USA, 2004; 101: 9607-9611).

The ATDC5 cell line was originally isolated from a differentiatingculture of AT805 teratocarcinoma. ATDC5 cells express a fibroblasticcell phenotype in a growing phase. Further information on the ATDC5 cellline can be found in Atsumi T, Miwa Y, Kimata K, Ikawa Y. A chondrogeniccell line derived from a differentiating culture of AT805teratocarcinoma cells. Cell Differ Dev. 1990; 30: 109-16.

The progenitor cells may be a population of cells enriched forStro-1^(bri). The progenitor cells may be homogeonous Stro-1^(bri)cells, or Stro-1^(bri) progeny cells.

Stro-1^(bri) cells are cells found in bone marrow, blood, dental pulpcells, adipose tissue, skin, spleen, pancreas, brain, kidney, liver,heart, retina, brain, hair follicles, intestine, lung, lymph node,thymus, bone, ligament, tendon, skeletal muscle, dermis, and periosteum;and are typically capable of differentiating into germ lines such asmesoderm and/or endoderm and/or ectoderm. Thus, Stro-1^(bri) cells arecapable of differentiating into a large number of cell types including,but not limited to, adipose, osseous, cartilaginous, elastic, muscular,and fibrous connective tissues. The specific lineage-commitment anddifferentiation pathway which these cells enter depends upon variousinfluences from mechanical influences and/or endogenous bioactivefactors, such as growth factors, cytokines, and/or localmicroenvironmental conditions established by host tissues. Stro-1^(bri)cells may thus be non-hematopoietic progenitor cells which divide toyield daughter cells that are either stem cells or are precursor cellswhich in time will irreversibly differentiate to yield a phenotypiccell.

In a further embodiment, the Stro-1^(bri) cells are enriched from asample obtained from a subject, e.g., a subject to be treated or arelated subject or an unrelated subject (whether of the same species ordifferent). The terms ‘enriched’, ‘enrichment’ or variations thereof areused herein to describe a population of cells in which the proportion ofone particular cell type or the proportion of a number of particularcell types is increased when compared with the untreated population.

In a further embodiment, the cells used in the present invention expressone or more markers individually or collectively selected from the groupconsisting of TNAP⁺, VCAM-1⁺, THY-1⁺, STRO-2⁺, CD45⁺, CD146⁺, 3G5⁺ orany combination thereof.

By “individually” is meant that the invention encompasses the recitedmarkers or groups of markers separately, and that, notwithstanding thatindividual markers or groups of markers may not be separately listedherein the accompanying claims may define such marker or groups ofmarkers separately and divisibly from each other.

By “collectively” is meant that the invention encompasses any number orcombination of the recited markers or groups of peptides, and that,notwithstanding that such numbers or combinations of markers or groupsof markers may not be specifically listed herein the accompanying claimsmay define such combinations or sub-combinations separately anddivisibly from any other combination of markers or groups of markers.

In one embodiment, the Stro-1^(bri) cells are additionally one or moreof TNAP⁺, VCAM-1⁺, THY-1⁺, STRO-2⁺ and/or CD146⁺.

A cell that is referred to as being “positive” for a given marker it mayexpress either a low (lo or dim) or a high (bright, bri) level of thatmarker depending on the degree to which the marker is present on thecell surface, where the terms relate to intensity of fluorescence orother marker used in the sorting process of the cells. The distinctionof lo (or dim or dull) and bri will be understood in the context of themarker used on a particular cell population being sorted. A cell that isreferred to as being “negative” for a given marker is not necessarilycompletely absent from that cell. This terms means that the marker isexpressed at a relatively very low level by that cell, and that itgenerates a very low signal when detectably labelled or is undetectableabove background levels.

The term “bright”, when used herein, refers to a marker on a cellsurface that generates a relatively high signal when detectablylabelled. Whilst not wishing to be limited by theory, it is proposedthat “bright” cells express more of the target marker protein (forexample the antigen recognised by STRO-1) than other cells in thesample. For instance, STRO-1^(bri) cells produce a greater fluorescentsignal, when labelled with a FITC-conjugated STRO-1 antibody asdetermined by fluorescence activated cell sorting (FACS) analysis, thannon-bright cells (STRO-1^(dull/dim)). In one embodiment, “bright” cellsconstitute at least about 0.1% of the most brightly labelled bone marrowmononuclear cells contained in the starting sample. In otherembodiments, “bright” cells constitute at least about 0.1%, at leastabout 0.5%, at least about 1%, at least about 1.5%, or at least about2%, of the most brightly labelled bone marrow mononuclear cellscontained in the starting sample. In a further embodiment,STRO-1^(bright) cells have 2 log magnitude higher expression of STRO-1surface expression relative to “background”, namely cells that areSTRO-Y. By comparison, STRO-1^(dim) and/or STRO-1^(inertmediate) cellshave less than 2 log magnitude higher expression of STRO-1 surfaceexpression, typically about 1 log or less than “background”.

As used herein the term “TNAP” is intended to encompass all isoforms oftissue non-specific alkaline phosphatase. For example, the termencompasses the liver isoform (LAP), the bone isoform (BAP) and thekidney isoform (KAP). In a further embodiment, the TNAP is BAP. In afurther embodiment, TNAP as used herein refers to a molecule which canbind the STRO-3 antibody produced by the hybridoma cell line depositedwith ATCC on 19 Dec. 2005 under the provisions of the Budapest Treatyunder deposit accession number PTA-7282.

Furthermore, in a further embodiment, the Stro-1^(bri) cells are capableof giving rise to clonogenic CFU-F.

In one embodiment, a significant proportion of the multipotential cellsare capable of differentiation into at least two different germ lines.Non-limiting examples of the lineages to which the multipotential cellsmay be committed include bone precursor cells; hepatocyte progenitors,which are multipotent for bile duct epithelial cells and hepatocytes;neural restricted cells, which can generate glial cell precursors thatprogress to oligodendrocytes and astrocytes; neuronal precursors thatprogress to neurons; precursors for cardiac muscle and cardiomyocytes,glucose-responsive insulin secreting pancreatic beta cell lines. Otherlineages include, but are not limited to, odontoblasts, dentin-producingcells and chondrocytes, and precursor cells of the following: retinalpigment epithelial cells, fibroblasts, skin cells such as keratinocytes,dendritic cells, hair follicle cells, renal duct epithelial cells,smooth and skeletal muscle cells, testicular progenitors, vascularendothelial cells, tendon, ligament, cartilage, adipocyte, fibroblast,marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericyte,vascular, epithelial, glial, neuronal, astrocyte and oligodendrocytecells.

In another embodiment, the Stro-1^(bri) cells are not capable of givingrise, upon culturing, to hematopoietic cells.

In one embodiment, the cells are taken from the subject to be treated,cultured in vitro using standard techniques and used to obtain expandedcells for administration to the subject as an autologous or allogeneiccomposition. In an alternative embodiment, cells of one or more of theestablished human cell lines are used. In another useful embodiment ofthe invention, cells of a non-human animal (or if the patient is not ahuman, from another species) are used.

The present invention also contemplates use of supernatant or solublefactors obtained or derived from Stro-1^(bri) cells and/or progeny cellsthereof (the latter also being referred to as expanded cells) which areproduced from in vitro culture in combination with the progenitor cells.Expanded cells of the invention may a have a wide variety of phenotypesdepending on the culture conditions (including the number and/or type ofstimulatory factors in the culture medium), the number of passages andthe like. In certain embodiments, the progeny cells are obtained afterabout 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9,or about 10 passages from the parental population. However, the progenycells may be obtained after any number of passages from the parentalpopulation.

The progeny cells may be obtained by culturing in any suitable medium.The term “medium”, as used in reference to a cell culture, includes thecomponents of the environment surrounding the cells. Media may be solid,liquid, gaseous or a mixture of phases and materials. Media includeliquid growth media as well as liquid media that do not sustain cellgrowth. Media also include gelatinous media such as agar, agarose,gelatin and collagen matrices. Exemplary gaseous media include thegaseous phase that cells growing on a petri dish or other solid orsemisolid support are exposed to. The term “medium” also refers tomaterial that is intended for use in a cell culture, even if it has notyet been contacted with cells. In other words, a nutrient rich liquidprepared for bacterial culture is a medium. A powder mixture that whenmixed with water or other liquid becomes suitable for cell culture maybe termed a “powdered medium”.

In an embodiment, progeny cells useful for the methods of the inventionare obtained by isolating TNAP⁺ STRO-1⁺ multipotential cells from bonemarrow using magnetic beads labelled with the STRO-3 antibody, and thenculture expanding the isolated cells (see Gronthos et al. Blood 85:929-940, 1995 for an example of suitable culturing conditions).

In one embodiment, such expanded cells (progeny) (optionally at leastafter 5 passages) can be TNAP⁻, CC9⁺, HLA class 1⁺, HLA class II⁻,CD14⁻, CD19⁻, CD3⁻, CD11a⁻c⁻, CD31⁻, CD86⁻, CD34⁻ and/or CD80⁻. However,it is possible that under different culturing conditions to thosedescribed herein that the expression of different markers may vary.Also, whilst cells of these phenotypes may predominate in the expendedcell population it does not mean that there is a minor proportion of thecells do not have this phenotype(s) (for example, a small percentage ofthe expanded cells may be CC9⁻). In one embodiment, expanded cells stillhave the capacity to differentiate into different cell types.

In one embodiment, an expended cell population used to obtainsupernatant or soluble factors, or cells per se, comprises cells whereinat least 25%, in a further embodiment at least 50%, of the cells areCC9+.

In another embodiment, an expanded cell population used to obtainsupernatant or soluble factors, or cells per se, comprises cells whereinat least 40%, in a further embodiment at least 45%, of the cells areSTRO-1⁺.

In a further embodiment, the expanded cells may express one or moremarkers collectively or individually selected from the group consistingof LFA-3, THY-1, VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, 3G5,CD49a/CD49b/CD29, CD49c/CD29, CD49d/CD29, CD 90, CD29, CD18, CD61,integrin beta 6-19, thrombomodulin, CD10, CD13, SCF, PDGF-R, EGF-R,IGF1-R, NGF-R, FGF-R, Leptin-R (STRO-2=Leptin-R), RANKL, STRO-1^(bright)and CD146 or any combination of these markers.

In one embodiment, progeny cells derived from STRO-1^(bri) cells arepositive for the marker Stro-1^(dim). These cells are referred to asTissue Specific Committed Cells (TSCCs) and are more committed todifferentiation than STRO-1^(bri) cells are therefore less able torespond inductive factors. Non-limiting examples of the lineages towhich TSCCs may be committed include hepatocyte progenitors, which arepluripotent for bile duct epithelial cells and hepatocytes; neuralrestricted cells, which can generate glial cell precursors that progressto oligodendrocytes and astrocytes, and neuronal precursors thatprogress to neurons; precursors for cardiac muscle and cardiomyocytes,glucose-responsive insulin secreting pancreatic beta cell lines. Othercommitted precursor cells include but are not limited to chondrocytes,osteoblasts, odontoblast, dentin-producing and chondrocytes, andprecursor cells of the following: retinal pigment epithelial cells,fibroblasts, skin cells such as keratinocytes, dendritic cells, hairfollicle cells, renal duct epithelial cells, smooth and skeletal musclecells, testicular progenitors, vascular endothelial cells, tendon,ligament, cartilage, adipocyte, fibroblast, marrow stroma, osteoclastand haemopoietic-supportive stroma, cardiac muscle, smooth muscle,skeletal muscle, pericyte, vascular, epithelial, glial, neuronal,astrocyte and oligodendrocyte cells. Precursors include those thatspecifically can lead to connective tissue particularly includingadipose, areolar, osseous, cartilaginous, elastic and fibrous connectivetissues.

In another embodiment, the progeny cells are Multipotential ExpandedSTRO-1⁺ Multipotential cells Progeny (MEMPs) as defined and/or describedin WO 2006/032092. Methods for preparing enriched populations of STRO-1⁺multipotential cells from which progeny may be derived are described inWO 01/04268 and WO 2004/085630. In an in vitro context STRO-1⁺multipotential cells will rarely be present as an absolutely purepreparation and will generally be present with other cells that aretissue specific committed cells (TSCCs). WO 01/04268 refers toharvesting such cells from bone marrow at purity levels of about 0.1% to90%. The population comprising progenitor cells from which progeny arederived may be directly harvested from a tissue source, or alternativelyit may be a population that has already been expanded ex vivo.

For example, the progeny may be obtained from a harvested, unexpanded,population of substantially purified STRO-1⁺ multipotential cells,comprising at least about 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80 or95% of total cells of the population in which they are present. Thislevel may be achieved, for example, by selecting for cells that arepositive for at least one marker individually or collectively selectedfrom the group consisting of TNAP, STRO-1^(bright), 3G5⁺, VCAM-1, THY-1,CD146 and STRO-2.

MEMPS can be distinguished from freshly harvested Stro-1^(bri) cells inthat they are positive for the marker STRO-1^(bri) and negative for themarker Alkaline phosphatase (ALP). In contrast, freshly isolatedStro-1^(bri) cells are positive for both STRO-1^(bri) and ALP. In afurther embodiment of the present invention, at least 15%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or 95% of the administered cells have thephenotype STRO-1^(bri) , ALP⁻. In a further embodiment the MEMPS arepositive for one or more of the markers Ki67, CD44 and/or CD49c/CD29,VLA-3, α3β1. In yet a further embodiment the MEMPs do not exhibit TERTactivity and/or are negative for the marker CD18.

The Stro-1^(bri) cell starting population may be derived from any one ormore tissue types including bone marrow, dental pulp cells, adiposetissue and skin, or perhaps more broadly from adipose tissue, teeth,dental pulp, skin, liver, kidney, heart, retina, brain, hair follicles,intestine, lung, spleen, lymph node, thymus, pancreas, bone, ligament,bone marrow, tendon and skeletal muscle.

It will be understood that in performing the present invention,separation of cells carrying any given cell surface marker can beeffected by a number of different methods, however, certain methods relyupon binding a binding agent (e.g., an antibody or antigen bindingfragment thereof) to the marker concerned followed by a separation ofthose that exhibit binding, being either high level binding, or lowlevel binding or no binding. The most convenient binding agents areantibodies or antibody-based molecules, preferably being monoclonalantibodies or based on monoclonal antibodies because of the specificityof these latter agents. Antibodies can be used for both steps, howeverother agents might also be used, thus ligands for these markers may alsobe employed to enrich for cells carrying them, or lacking them.

The antibodies or ligands may be attached to a solid support to allowfor a crude separation. The separation techniques preferably maximisethe retention of viability of the fraction to be collected. Varioustechniques of different efficacy may be employed to obtain relativelycrude separations. The particular technique employed will depend uponefficiency of separation, associated cytotoxicity, ease and speed ofperformance, and necessity for sophisticated equipment and/or technicalskill. Procedures for separation may include, but are not limited to,magnetic separation, using antibody-coated magnetic beads, affinitychromatography and “panning” with antibody attached to a solid matrix.Techniques providing accurate separation include but are not limited toFACS. Methods for performing FACS will be apparent to the skilledartisan.

Antibodies against each of the markers described herein are commerciallyavailable (e.g., monoclonal antibodies against STRO-1 are commerciallyavailable from R&D Systems, USA), available from ATCC or otherdepositary organization and/or can be produced using art recognizedtechniques.

It is preferred that the method for isolating Stro-1^(bri) cells, forexample, comprises a first step being a solid phase sorting steputilising for example magnetic activated cell sorting (MACS) recognisinghigh level expression of STRO-1. A second sorting step can then follow,should that be desired, to result in a higher level of precursor cellexpression. This second sorting step might involve the use of two ormore markers. The method obtaining Stro-1^(bri) cells might also includethe harvesting of a source of the cells before the first enrichment stepusing known techniques. Thus the tissue will be surgically removed.Cells comprising the source tissue will then be separated into a socalled single cells suspension. This separation may be achieved byphysical and or enzymatic means.

Once a suitable Stro-1^(bri) cell population has been obtained, it maybe cultured or expanded by any suitable means to obtain MEMPs.

In one embodiment, the cells are taken from the subject to be treated,cultured in vitro using standard techniques and used to obtainsupernatant or soluble factors or expanded cells for administration tothe subject as an autologous or allogeneic composition. In analternative embodiment, cells of one or more of the established humancell lines are used to obtain the supernatant or soluble factors. Inanother useful embodiment of the invention, cells of a non-human animal(or if the patient is not a human, from another species) are used toobtain supernatant or soluble factors.

The invention can be practised using cells from any non-human animalspecies, including but not limited to non-human primate cells, ungulate,canine, feline, lagomorph, rodent, avian, and fish cells. Primate cellswith which the invention may be performed include but are not limited tocells of chimpanzees, baboons, cynomolgus monkeys, and any other New orOld World monkeys. Ungulate cells with which the invention may beperformed include but are not limited to cells of bovines, porcines,ovines, caprines, equines, buffalo and bison. Rodent cells with whichthe invention may be performed include but are not limited to mouse,rat, guinea pig, hamster and gerbil cells. Examples of lagomorph specieswith which the invention may be performed include domesticated rabbits,jack rabbits, hares, cottontails, snowshoe rabbits, and pikas. Chickens(Gallus gallus) are an example of an avian species with which theinvention may be performed.

Cells useful for the methods of the invention may be stored before use,or before obtaining the supernatant or soluble factors. Methods andprotocols for preserving and storing of eukaryotic cells, and inparticular mammalian cells, are known in the art (cf., for example,Pollard, J. W. and Walker, J. M. (1997) Basic Cell Culture Protocols,Second Edition, Humana Press, Totowa, N.J.; Freshney, R. I. (2000)Culture of Animal Cells, Fourth Edition, Wiley-Liss, Hoboken, N.J.).

Genetically-Modified Cells

In one embodiment, the Stro-1^(bri) cells and/or progeny cells thereofare genetically modified, e.g., to express and/or secrete a protein ofinterest, e.g., a protein providing a therapeutic and/or prophylacticbenefit, e.g., insulin, glucagon, somatostatin, trypsinogen,chymotrypsinogen, elastase, carboxypeptidase, pancreatic lipase oramylase or a polypeptide associated with or causative of enhancedangiogenesis or a polypeptide associated with differentiation of a cellinto a pancreatic cell or a vascular cell.

Methods for genetically modifying a cell will be apparent to the skilledartisan. For example, a nucleic acid that is to be expressed in a cellis operably-linked to a promoter for inducing expression in the cell.For example, the nucleic acid is linked to a promoter operable in avariety of cells of a subject, such as, for example, a viral promoter,e.g., a CMV promoter (e.g., a CMV-IE promoter) or a SV-40 promoter.Additional suitable promoters are known in the art and shall be taken toapply mutatis mutandis to the present embodiment of the invention.

In one embodiment, the nucleic acid is provided in the form of anexpression construct. As used herein, the term “expression construct”refers to a nucleic acid that has the ability to confer expression on anucleic acid (e.g. a reporter gene and/or a counter-selectable reportergene) to which it is operably connected, in a cell. Within the contextof the present invention, it is to be understood that an expressionconstruct may comprise or be a plasmid, bacteriophage, phagemid, cosmid,virus sub-genomic or genomic fragment, or other nucleic acid capable ofmaintaining and/or replicating heterologous DNA in an expressibleformat.

Methods for the construction of a suitable expression construct forperformance of the invention will be apparent to the skilled artisan andare described, for example, in Ausubel et al (In: Current Protocols inMolecular Biology. Wiley Interscience, ISBN 047 150338, 1987) orSambrook et al (In: Molecular Cloning: Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).For example, each of the components of the expression construct isamplified from a suitable template nucleic acid using, for example, PCRand subsequently cloned into a suitable expression construct, such asfor example, a plasmid or a phagemid.

Vectors suitable for such an expression construct are known in the artand/or described herein. For example, an expression vector suitable forthe method of the present invention in a mammalian cell is, for example,a vector of the pcDNA vector suite supplied by Invitrogen, a vector ofthe pCI vector suite (Promega), a vector of the pCMV vector suite(Clontech), a pM vector (Clontech), a pSI vector (Promega), a VP 16vector (Clontech) or a vector of the pcDNA vector suite (Invitrogen).

The skilled artisan will be aware of additional vectors and sources ofsuch vectors, such as, for example, Invitrogen Corporation, Clontech orPromega.

Means for introducing the isolated nucleic acid molecule or a geneconstruct comprising same into a cell for expression are known to thoseskilled in the art. The technique used for a given organism depends onthe known successful techniques. Means for introducing recombinant DNAinto cells include microinjection, transfection mediated byDEAE-dextran, transfection mediated by liposomes such as by usinglipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA),PEG-mediated DNA uptake, electroporation and microparticle bombardmentsuch as by using DNA-coated tungsten or gold particles (Agracetus Inc.,WI, USA) amongst others.

Alternatively, an expression construct of the invention is a viralvector. Suitable viral vectors are known in the art and commerciallyavailable. Conventional viral-based systems for the delivery of anucleic acid and integration of that nucleic acid into a host cellgenome include, for example, a retroviral vector, a lentiviral vector oran adeno-associated viral vector. Alternatively, an adenoviral vector isuseful for introducing a nucleic acid that remains episomal into a hostcell. Viral vectors are an efficient and versatile method of genetransfer in target cells and tissues. Additionally, high transductionefficiencies have been observed in many different cell types and targettissues.

For example, a retroviral vector generally comprises cis-acting longterminal repeats (LTRs) with packaging capacity for up to 6-10 kb offoreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of a vector, which is then used to integratethe expression construct into the target cell to provide long termexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simianimmunodeficiency virus (SrV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J Virol. 56:2731-2739(1992); Johann et al, J. Virol. 65:1635-1640 (1992); Sommerfelt et al,Virol. 76:58-59 (1990); Wilson et al, J. Virol. 63:274-2318 (1989);Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700; Miller andRosman BioTechniques 7:980-990, 1989; Miller, A. D. Human Gene Therapy7:5-14, 1990; Scarpa et al Virology 75:849-852, 1991; Burns et al. Proc.Natl. Acad. Sci USA 90:8033-8037, 1993).

Various adeno-associated virus (AAV) vector systems have also beendeveloped for nucleic acid delivery. AAV vectors can be readilyconstructed using techniques known in the art. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 andWO 93/03769; Lebkowski et al. Molec. Cell. Biol. 5:3988-3996, 1988;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter Current Opinion in Biotechnology 5:533-539, 1992; Muzyczka.Current Topics in Microbiol, and Immunol. 158:97-129, 1992; Kotin, HumanGene Therapy 5:793-801, 1994; Shelling and Smith Gene Therapy 7:165-169,1994; and Zhou et al. J Exp. Med. 179:1867-1875, 1994.

Additional viral vectors useful for delivering an expression constructof the invention include, for example, those derived from the pox familyof viruses, such as vaccinia virus and avian poxvirus or an alphavirusor a conjugate virus vector (e.g. that described in Fisher-Hoch et al.,Proc. Natl Acad. Sci. USA 56:317-321, 1989).

In a further embodiment, the progenitor cells are a population of cellsenriched for Stro-3^(bri), or homogeonous Stro-3^(bri) cells, orStro-3^(bri) progeny cells.

Polysulfated Polysaccharides

The present invention also relates to the use of polysulfatedpolysaccharide compounds. The polysulfated polysaccharide family can beconsidered to be any naturally occurring or semi-synthetic/syntheticpolysulfated polysaccharide or a biologically active fragment thereofthat contains two or more sugar rings or carbohydrate structures towhich one or more sulfate ester groups are covalently attached asexemplified by heparin and pentosan polysulfate.

Examples of polysulfated polysaccharides falling within the scope of thepresent invention are naturally occurring high molecular weight heparin,low molecular weight heparins, heparan sulfate, pentosan polysulfate,chondroitin polysulfate, chitosan polysulfate, dermatan polysulfatesulodexide, dextran polysulfate, polysulfated inulin, sulfatedlactobionic acid amide, sulfated bis-aldonic acid amide, sucroseoctasulfate, fucoidan-1, fucoidan-2, sulfated beta-cyclodextrin,sulfated gamma-cyclodextrin and small sulfated compounds including, butare not limited to, inositol hexasulfate.

A specific list of polysulfated polysaccharides are pentosanpolysulfate, calcium pentosan polysulfate, magnesium pentosanpolysulfate, sodium pentosan polysulfate, polysulfated chondroitin anddextran polysulfate.

Further examples of the polysaccharides suitable for the presentinvention include polysulfated polysaccharides, polysulfated dextran,polysulfated cyclodextran, polysulfated chondroitin, and pentosanpolysulfate as its alkali metal or alkaline earth metal salt, forexample its calcium or sodium salt, transition metals such as copper andzinc and noble metals such as platinum. Further examples arepolysulfated polysaccharide derivatives of homopolysaccharides orheteropolysaccharides, which can be linear or branched. The sugars maycome from but are not limited to pentoses or hexoses such as galactose,mannose, glucose, rhanose, fructose, sorbose, xylose, D-arabinose,ribose, L-arabinose, glucuronic acid and their derivatives.

The present invention also encompasses biologically active molecularfragments of polysulfated polysaccharides or analogues or derivatives ofpolysulfated polysaccharides.

One polysulfated polysaccharide is pentosan polysulfate (PPS). The basicstructure of PPS consists of pentoses, i. e. (1→4) linkedbeta-D-xylopyranose units containing glucuronic acid groups atstatistically every 10th unit.

Shown below is the structural formula of pentosan polysulfate (PPS)isolated from beechwood hemicellulose (Fagus silvatica). This formulashows that the linear xylan (pentosan) backbone of pentosan polysulfatecontains on average one 4-O-methyl-glucuronate side chain linked to the2-position on every tenth xylose (pentose) ring.

The calcium and magnesium derivatives of PPS (CaPPS or MgPPS) is whenR═SO₃—Ca⁺½ or Mg⁺½. The sodium derivative is when R═SO₃—Na.

Pentosan polysulfate as its calcium or sodium salt has an averagemolecular weight of about 5700 Daltons and a sulphur content of about16%. This compound has been known since the early 1960s to be asynthetic heparinoid and an anti-thrombotic agent.

The particular complexing ions may be selected from the group consistingof the alkali metals, e. g. Na⁺, and K⁺, alkaline earth metals, e. g.Ca²⁺, Zn²⁺, Mg²⁺, Ba²⁺, as well as Ag⁺, Au⁺, Pb²⁺, Cu²⁺, Au²⁺, Pd²⁺,Pd⁴⁺, Pt⁴⁺, Pt²⁺, trivalent metal ions, and quaternary ammonium compoundcomplexes. Examples of the latter compounds are pyridinium chloride,tetraalkyl ammonium chloride, choline chloride, cetylpyridiniumchloride, N-cetyl-N,N,N-trialkylammonium chloride or their derivatives.In one particular embodiment, the calcium complex is used.

Preparation of the polysulfate polysaccharide-metal complexes isdescribed in detail in U.S. Pat. No. 5,668,116, the entire disclosure ofwhich is incorporated herein by reference.

Further information relating to polysulfate polysaccharides and PPS canbe found in WO02/41901, the entire disclosure of which is incorporatedherein by reference. Further information can also be found in SeminArthritis Rheum. 1999 February; 28(4):211-67 Ghosh—The pathobiology ofosteoarthritis and the rationale for the use of pentosan polysulfate forits treatment, the entire disclosure of which is incorporated herein byreference.

In one embodiment, the polysulfated polysaccharide is pentosanpolysulfate sodium. An example of this is SP54, manufactured by BenePharmachem, Germany, which is a polysaccharide, esterified withsulphuric acid, more specifically a pentosan polysulfate sodium. Thesemisynthetic production of pentosan polysulfate sodium assuresconsistent and reproducible manufacturing with a defined range ofmolecular weight (4000 to 6000 daltons).

A further polysulfated polysaccharide is polysulfated chondroitin. Anexample of this is Arteparon® (trade mark of Luitpold-Werk) whichconsists predominantly of polysulfated chondroitin. It has been used asan antiarthritic drug. More specifically, it is a heterogeneoussemi-synthetic glycosaminoglycan polysulfate in which the predominant(about 93%) disaccharide repeating unit is hexuronic acid glycosidicallylinked to galactosamine. Approximately four of the free hydroxyl groupsof the disaccharide repeating unit of Arteparon are esterified bysulfate groups to give a sulphur content of about 13.0% by weight. Thecommercial preparation has a molecular weight of about 10,000 Daltons.

Preparation of the polysulfate polysaccharide-metal complexes isdescribed in detail in U.S. Pat. No. 5,668,116, the entire disclosure ofwhich is incorporated herein by reference.

Further information relating to polysulfate polysaccharides and PPS canbe found in WO02/41901, the entire disclosure of which is incorporatedherein by reference. Further information can be found in Semin ArthritisRheum. 1999 February; 28(4):211-67 Ghosh—The pathobiology ofosteoarthritis and the rationale for the use of pentosan polysulfate forits treatment, the entire disclosure of which is incorporated herein byreference.

In particular, the methods of manufacture, isolation and purificationtogether with suitable carriers compositions and formulations areincorporated into the present application.

Further information can also be found in Ghosh P, Edelman J, March L andSmith M. Effects of pentosan polysulfate in osteoarthritis of the knee:A randomised, double blind, placebo-controlled pilot study. CurrentTherapeutic Research, 2005, 66: 552-571, and which provides informationon an OA clinical study using PPS given by intra-muscular injection. Theinformation on PPS, and the methods used in the trial including thedosage regimes and administration methods are hereby incorporated byreference into the present application.

Non-Collagenous NC4 Domain of Alpha IX Collagen

According to a further embodiment, the polypeptide chosen is anon-collagenous NC4 domain of alpha IX collagen or a biologically activemolecular fragment thereof. Specific information about suitable NC4domain polypeptides can be found in International applicationPCT/AU2004/000788, the contents of which are incorporated in theirentirety.

In particular, PCT/AU2004/000788 discloses a number of polypeptide andtheir sequences that can be used in the present invention together withways of expressing and purifying the polypeptides. Thus,PCT/AU2004/000788 includes examples of suitable NC4 domain polypeptidesand ways of making them which are specifically incorporated into thepresent specification. In particular, the amino acid sequences as setout in page 7 lines 24 to page 8 line 6 together with the sequences asset out in FIGS. 1-7 are specifically incorporated into the presentapplication. Furthermore, the methods of recovering polypeptides as setout in page 9 line 10 to page 10 line 36 are specifically incorporatedinto the present application. The autolysis techniques set out in thedetailed description from page 15 together with the separating andrecovery steps set out in the detailed description from page 18 andspecifically the polypeptides as set out on pages 20-24 are incorporatedinto the present invention. Finally, the partial amino acid sequence ofFIG. 7 is incorporated into the present application.

As said above, the NC4 domain is discussed in PCT/AU2004/000788. Itincludes the complete amino acid sequence predicted from the genesequence and obtained by expression in E. coli (see Table 1).

However, it should be noted that the protein products obtained byexpression using K. lactis consisted of the full length human NC4 plus atruncated form (MW=24 kDa), with both forms being glycosylated, theseare also included in Table 1 and there preparation and ID are describedin the methods section. Both the E. coli and K. lactis expressedproteins were evaluated in animal models and in vitro assays and may beidentified by the codes AWR-01 and PBA-1200P respectively. Theprogenitor cells used in these experiments was the mouse ATDC5 which iscommercially available as discussed in the methods section. Thedifferentiated cells used included: chondrocytes from human jointcartilage, normal ovine and porcine cartilage and chondrocytes andsynovial fibroblasts derived from synovial tissue from OA patientsundergoing total joint replacement surgery.

In particular, the following sequences also define polypeptides for usein the present application.

TABLE 1Amino acid sequences of interest but shown without N or O glycosylationSEQ ID NO: 1 1) hNC4: (Full length human NC4 without signal peptide)        10         20         30         40         50         60AVKRRPRFPV NSNSNGGNEL CPKIRIGQDD LPGFDLISQF QVDKAASRRA IQRVVGSATL        70         80         90        100        110        120QVAYKLGNNV DFRIPTRNLY PSGLPEEYSF LTTFRMTGST LKKNWNIWQI QDSSGKEQVG       130        140        150        160        170        180IKINGQTQSV VFSYKGLDGS LQTAAFSNLS SLFDSQWHKI MIGVERSSAT LFVDCNRIES       190        200        210        220        230        240LPIKPRGPID IDGFAVLGKL ADNPQVSVPF ELQWMLIHCD PLRPRRETCH ELPARITPSQ TTDERSEQ ID NO: 22) Truncated hNC4 (residues 6-224) obtained during expression from K. lactis cultures by action of putative by proline endopeptidase:        10         20         30         40         50         60RFPVNSNSNG GNELCPKIRI GQDDLPGFDL ISQFQVDKAA SRRAIQRVVG SATLQVAYKL        70         80         90        100        110        120GNNVDFRIPT RNLYPSGLPE EYSFLTTFRM TGSTLKKNWN IWQIQDSSGK EQVGIKINGQ       130        140        150        160        170       180TQSVVFSYKG LDGSLQTAAF SNLSSLFDSQ WHKIMIGVER SSATLFVDCN RIESLPIKPR       190        200        210GPIDIDGFAV LGKLADNPQV SVPFELQWML IHCDPLRP3) Sequences underlined in bolded red which were identified by proteomics and described in Patent Application PCT/AU2004/000788  1 Met Lys Thr Cys Trp Lys Ile Pro Val Phe Phe Phe Val Cys Ser 16 Phe Leu Glu Pro Trp Ala Ser Ala  23 Ala Val Lys Arg Arg  Pro Arg 31  Phe Pro Val Asn Ser Asn Ser Asn Gly  Gly  Asn Glu Leu Cys Pro  46 Lys  Ile  Arg Ile Gly Gln Asp Asp Leu Pro Gly Phe Asp Leu Ile  61 Ser Gln Phe Gln  Val  Asp Lys Ala Ala Ser Arg Arg Ala Ile Gln  76 Arg Val Val Gly Ser  Ala Thr  Leu Gln Val Ala Tyr Lys Leu Gly  91 Asn Asn Val Asp Phe Arg  Ile Pro Thr Arg Asn Leu Tyr Pro Ser106 Gly Leu  Pro Glu Glu Tyr Ser Phe Leu Thr Thr Phe Arg Met Thr 121 Gly Ser Thr Leu  Lys  Lys  Asn  Trp  Asn  Ile Trp Gln Ile Gln Asp 136 Ser Ser Gly Lys Glu Gln Val Gly Ile Lys Ile Asn Gly Gln Thr 151 Gln Ser Val  Val  Phe Ser Tyr Lys Gly Leu Asp Gly Ser Leu Gln 166 Thr Ala Ala Phe Ser Asn Leu  Ser  Ser Leu Phe Asp Ser Gln Trp 181 His Lys Ile Met Ile Gly Val Glu Arg Ser Ser Ala Thr Leu Phe 196 Val Asp Cys Asn Arg Ile Glu Ser Leu Pro Ile Lys Pro  Arg Gly211 Pro Ile Asp Ile Asp Gly Phe Ala Val Leu Gly Lys Leu Ala Asp226 Asn Pro Gln Val Ser Val Pro Phe Glu Leu Gln Trp Met Leu Ile241 His Cys Asp Pro Leu Arg Pro Arg Arg Glu Thr Cys His Glu Leu256 Pro Ala Arg Ile Thr Pro Ser Gln Thr Thr Asp Glu Arg 268

A sequence composition between bovine human, murine and chick NC4sequences is provided in FIG. 37. Conserved sequences are as follows:

SEQ ID NO: 3 K/QSVSN/A/EFSYKG SEQ ID NO: 4 KI/LMIG/SVER/TS/TSEQ ID NO: 5 KLGNNVDFRI SEQ ID NO: 6 R/KINES/TLP/NIKPR/KG SEQ ID NO: 7KH/N/YWS/N/TIWQIQDS/AGK/R SEQ ID NO: 8 K/QSVSNFSYKG SEQ ID NO: 9KIMIGVERTS SEQ ID NO: 10 RIESLPIKPRG SEQ ID NO: 11 KH/NWS/NIWQIQDSGKSEQ ID NO: 12 RIGQDDLPGFDLISQFQI/VDKA SEQ ID NO: 13RH/NLYPN/SGLPEEYSFLTTFR SEQ ID NO: 14 FSNLP/SSLFDSQWHKI SEQ ID NO: 15RSSATLFVDCNRI SEQ ID NO: 16 SVSFSYKG SEQ ID NO: 17 KIMIGVERSSEQ ID NO: 18 KLGNNVDFRI SEQ ID NO: 19 RIESLPIKPRG SEQ ID NO: 20KHWSIWQIQDSSGK SEQ ID NO: 21 RIGQDDLPGFDLISQFQIDKA SEQ ID NO: 22RHLYPNGLPEEYSFLTTFRM SEQ ID NO: 23 FSNKOSKFDSQWHKI SEQ ID NO: 24RSSATLFVDCNRI

Cryopreservation

There are various techniques to cryopreserve progenitor cells known tothe person skilled in the art. One example procedure is as follows:

Examination:

Prior to freezing, the cells should be maintained in an actively growingstate to insure maximum health and a good recovery. Ideally, the culturemedium should be changed the previous day. Using an inverted microscope,quickly check the general appearance of the culture. Look for signs ofmicrobial contamination. It is also important to examine the culturewith the unaided eye to look for small fungal colonies that may befloating at the medium-air interface and thus not visible through themicroscope. It is best if the cultures are maintained antibiotic-freefor at least one week prior to freezing to help uncover any cryptic(hidden) culture contaminants.

Cell Harvesting and Freezing:

Treat the cells gently during harvesting since it is very difficult forcells damaged during harvesting to survive the additional damage thatoccurs during the freezing and thawing processes. You should be able toobtain up to 1.5×107 cells from a near confluent T-75 flask (dependingon cell type and degree of confluency). This should be enough cells toset up at least several vials at 2×10⁶ cells/vial.

1. Using a sterile pipette, remove and discard the old culture medium.

2. For a T-75 flask, rinse the cell monolayer with 5 mL of calcium- andmagnesium-free phosphate buffered saline (CMF-PBS) to remove all tracesof foetal bovine serum.

3. Add 4 to 5 mL of the trypsin solution (in CMF-PBS) to the flask andallow cells to incubate for at least one minute. (Prewarming of theenzyme 4 solution will decrease the exposure period.) Withdraw about 3mL of the trypsin solution and allow the cells to round up and loosen.

4. Check the progress of the enzyme treatment every few minutes on aninverted phase contrast microscope. Once all of the cells have roundedup, gently tap the flask to detach them from the plastic surface. Thenadd 5 mL of growth medium to the cell suspension and, using the samepipette, vigorously wash any remaining cells from the bottom of theculture vessel.

5. Collect the suspended cells in a 15 mL centrifuge tube and place onice. Take a sample for counting and then spin at 100×g for 5 minutes toobtain a cell pellet. While the cells are spinning, do a viable cellcount (with the trypan blue solution) and calculate the number ofcells/mL and the total cell number.

6. Remove the supernatant from the centrifuged cells and resuspend thecell pellet in enough of the cryoprotective medium containing 10% DMSO(DMSO is most often used at a final concentration of 5 to 15% to give afinal cell concentration of 1 to 2×106 cells/mL.

7. Label the appropriate number of cryogenic vials with the cell line,and the date. Then add 1.5 to 1.8 mL of the DMSO containing cellsuspension to each of the vials and seal.

8. Place the vials in the controlled rate freezer overnight. After 24hours, the cells should be transferred to a liquid nitrogen freezer forpermanent storage.

9. Record the appropriate information about the cells in your cellrepository records. Fully detail in these records the culture's storageconditions, including all of the following information: cultureidentity, passage or population doubling level, date frozen, freezingmedium and method used, number of cells per vial, total number of vialsinitially frozen and the number remaining, their locations, theirexpected viability and results of all quality control tests performed(sterility, mycoplasma, species, karyotype, etc.). Additional cultureinformation, especially its origin, history, growth parameters, specialcharacteristics and applications, is also helpful and should be includedwhenever possible.

Cell Thawing and Recovery:

1. Using appropriate safety equipment, remove the vial from its storagelocation and carefully check both the label and storage record to ensurethat it is the correct culture. Place the vessel in warm water,agitating gently until completely thawed. Rapid thawing (60 to 90seconds at 37° C.) provides the best recovery for most cell cultures; itreduces or prevents the formation of damaging ice crystals within cellsduring rehydration.

2. Since some cryoprotective agents may damage cells upon prolongedexposure, remove the agents as quickly and gently as possible. Severalapproaches are used depending on both the cryoprotective agents andcharacteristics of the cells:

-   -   a) Most cells recover normally if they have the cryoprotective        agent removed by a medium change within 6 to 8 hours of thawing.        Transfer the contents of the ampule or vial to a T-75 flask or        other suitable vessel containing 15 to 20 mL of culture medium        and incubate normally. As soon as a majority of the cells have        attached (usually 3 to 4 hours), remove the medium containing        the now diluted cryoprotective agent and replace with fresh        medium.    -   b) For cells that are sensitive to cryoprotective agents,        removing the old medium is easily accomplished by gentle        centrifugation. Transfer the contents of the vial or ampule to a        15mL centrifuge tube containing 10 mL of fresh medium and spin        for 5 minutes at 100×g. Discard the supernatant containing the        cryoprotective agent and resuspend the cell pellet in fresh        medium. Then transfer the cell suspension to a suitable culture        vessel and incubate normally.

Support Matrix

Recent advances in biology and material science have brought tissueengineering to the forefront of new cartilage repair techniques. Thecombination of autologous cells, specifically designed scaffolds,bioreactors, mechanical stimulations and growth factors offer promisingavenues for carilage tissue regeneration.

Bioscaffolds Mimic Extracellular Matrix.

Current tissue-engineering strategies provide scaffolds derived fromboth synthetic (e.g., polyglycolic acid) and naturally-derived (e.g.,collagen) materials to form the cell-scaffold construct. Currentlyavailable tissue scaffold products include small intestine submucosa(Restore™, porcine SIS, DePuy Orthopaedics), (CuffPatch™, porcine SIS,Organogenesis), (SIS; Cook Biotech, Inc.), reformulated collagenscaffolds (3D Collagen Composite, BD Biosciences), acellular humandermal collagen matrices (Graftjacket®, Wright Medical Technologies),fetal bovine dermis (TissueMend®, Stryker), and synthetic polymerscaffolds, primarily polyesters (e.g. PGA, PCL, and PLA).

Tissue engineered scaffolds that have recently been described includingcollagen scaffolds, chrondrocyte seeded scaffolds, articular chondrocyteseeded type II collagen-GAG scaffolds [Vickers et al, Tissue Eng. 2006May; 12(5): 1345-55] and composite scaffolds comprising polyethyleneoxide (PEO) and chitosan [Kuo Y C et al; J. Biomed Mater Res A. 2008Nov. 3] An alternate form described by Nettles D L et al., [Tissue Engpart A 2008 July; 14(7): 1133-40] is an injectable cross-linkableelastin-like polypeptide (ELP) gel for application to cartilage matrixrepair.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. Potential matrices for the compositions may bebiodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid and polyanhydrides. Otherpotential materials are biodegradable and biologically well defined,such as bone or dermal collagen. Further matrices are comprised of pureproteins or extracellular matrix components. Other potential matricesare nonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalcium phosphate. The bioceramics may be altered in composition,such as in calcium-aluminate-phosphate and processing to alter poresize, particle size, particle shape, and biodegradability.

In one embodiment, the scaffold is derived from a synthetic material. Inanother embodiment the scaffold is derived from naturallyderived-materials. Alternatively, the scaffold is a combination ofsynthetic and naturally derived materials.

In one embodiment, and as described in the examples, the support matrixis a collagen sponge. The composition of the invention including thecells, pentosan polysulfate optionally in combination with a NC4polypeptidecan be implanted or infused or perfused into the sponge.Since sponges can be delicate to implant, the sponge is optionallyinserted into a resorbable cage.

Compositions

In all cases, the compositions and formulations as discussed herein aresuitable for the polysaccharides and/or polypeptides of the presentinvention. In particular, the compositions and formulations are suitablefor polysulfated polysaccharides, in one embodiment pentosanpolysulfate, calcium pentosan polysulfate, magnesium pentosanpolysulfate and/or sodium pentosan polysulfated alone. The compositionsand formulations are also suitable for combinations of the compoundsdiscussed herein, for example combinations of polysaccharides and/orpolypeptides, and in particular combinations of NC4 domain polypeptidesand pentosan polysulfate and its salts.

According to the present invention, compositions comprising apolysaccharide and/or polypeptide as disclosed herein, particularlypolysulfated polysaccharides optionally with NC4 domain, in oneembodiment pentosan polysulfate, calcium pentosan polysulfate and/orsodium pentosan polysulfate or a fragment or truncated form are suitablefor human or animal usage in human and veterinary medicine and willtypically comprise any one or more of a pharmaceutically acceptablediluent, carrier or excipient. Acceptable carriers or diluents fortherapeutic use are well known in the pharmaceutical art, and aredescribed for example in Remington's Pharmaceutical Sciences MackPublishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceuticalcarrier excipient or diluent can be selected with regard to the intendedroute of administration and standard pharmaceutical practice. Thecompositions may comprise as, or in addition to, the carrier, excipientor diluent, any suitable binder, lubricant, suspending agent(liposomes), coating agent, or solubilising agent.

It is well known in the art that there may be differentcomposition/formulation requirements dependent on the different deliverysystems. For example, polysaccharides and/or proteins comprising a NC4domain can be dissolved in saline. Alternatively these compounds can bemade up in a solution provided with a two part liquid-powder that ismixed before use.

Implantable (subcutaneous) slow release capsules are often used forcontraceptives like Implanon™ or Depo-Provera™ injections and would beuseful for administration of a composition of the present invention.

In another example, the composition of the present invention may beformulated to be delivered using an implanted mini-pump wherein thecomposition is typically administered by continuous infusion into thedesired location.

In an alternate embodiment, compositions of the invention can beinjected or otherwise implanted parenterally for example, intravenously,subcutaneously, intra-muscularly, intra-articularly, intra-discally orintra-dermally. In a further embodiment the formulation is administeredsubcutaneously, intra-dermally or intra-articularly.

Subcutaneous and intra-dermal formulations can also contain one or moreadditional agents such as for example soft-tissue filler substances,lidocaine (local anaesthetic), matrix metalloproteinase inhibitors,antioxidants and anti-inflammatory agents (corticosteroids).

Various formulations for intra-dermal delivery of a drug may compriseone or more of the following ingredients: albumin, buffer, bufferedsaline, buffered salt solution, and anesthetic (preferably local).

The present invention extends to combination therapies for use in thetreatment of the diseases discussed herein. Particularly, in oneexample, the present invention extends to the use of a polysulfatedpolysaccharide and a polypeptide for use in the treatment of variousdegenerative conditions. It should be understood that these agents canbe administered at the same time or a different time. Thus thecombination therapy may comprise the active agents being administered atthe same time either in a single formulation or in multiple formulationsadministered at the same or different times. Equally, the combinationtherapy may comprise the active agents being administered in differentformulations at different times. The formulations could be administeredsequentially and may be separated by a period of time including hours,days, weeks and months.

Example formulations suitable for injection into an animal, for exampleintra-dermally, sub-cutaneously or intra-muscularly include:

1) A polysulfated polysaccharides, in one embodiment pentosanpolysulfate, calcium pentosan polysulfate and/or sodium pentosanpolysulfate together with progenitor cells dissolved in sterile water.

2) A polysulfated polysaccharides, in one embodiment pentosanpolysulfate, calcium pentosan polysulfate and/or sodium pentosanpolysulfated, together with progenitor cells, dissolved in 0.9% sterilesaline (150 mM NaCl); +/−human albumin (0.01%-0.5%); +/−localanaesthetic; examples e.g. bupivacaine hydrochloride (1.25-5 mg/ml);+/−adrenaline acid tartrate (0.0045-0.0091 mg/ml); +/−lidocaine(0.5-2%); +/−epinephrine (1:100,000-1:200,000).

3) A polysulfated polysaccharides, in one embodiment pentosanpolysulfate, calcium pentosan polysulfate and/or sodium pentosanpolysulfate, together with progenitor cells, dissolved in phosphatebuffered saline; +/−human albumin (0.01-0.5%); +/−local anaesthetic; 137mM NaCl; 2.7 mM KCl; 10 mM phosphate buffer; 150 mM NaCl; 150 mMNaH₂PO₄/Na₂HPO₄.

4) A polysulfated polysaccharides, in one embodiment pentosanpolysulfate, calcium pentosan polysulfate and/or sodium pentosanpolysulfated, together with progenitor cells, dissolved inphosphate-citrate buffer (50 mM) +/−sodium perborate (0.03%); +/−humanalbumin (0.01-0.5%); +/−local anaesthetics.

5) A polysulfated polysaccharides, in one embodiment pentosanpolysulfate, calcium pentosan polysulfate and/or sodium pentosanpolysulfate, together with progenitor cells, dissolved in a solution ofsterile water containing carboxymethylcellulose (2.7%) and mannitol.

6) A polysulfated polysaccharides, in one embodiment pentosanpolysulfate, calcium pentosan polysulfate and/or sodium pentosanpolysulfate, together with progenitor cells, incorporated intobiocompatible polyalkymide hydrogels (eg Bio-Alcamid® made by PolymekonS.r.l. (Milan, Italy).

7) A polysulfated polysaccharides, in one embodiment pentosanpolysulfate, calcium pentosan polysulfate and/or sodium pentosanpolysulfate, together with progenitor cells, incorporated into hylan Bgel (e.g. Hylaform® Plus).

8) A polysulfated polysaccharides, in one embodiment pentosanpolysulfate, calcium pentosan polysulfate and/or sodium pentosanpolysulfate, together with progenitor cells, incorporated intostabilised hyaluronic acid gel (e.g. Restylane®).

9) A polysulfated polysaccharides, in one embodiment pentosanpolysulfate, calcium pentosan polysulfate and/or sodium pentosanpolysulfate, together with progenitor cells, incorporated intopoly-L-lactic acid solution (e.g. Sculptra®).

10) A polysulfated polysaccharides, in one embodiment pentosanpolysulfate, calcium pentosan polysulfate and/or sodium pentosanpolysulfate, together with progenitor cells, incorporated dissolved in asolution of Krebs-Ringer's solution containing NaCl 118.1 mM; KCl 3.4mM; CaCl₂ 2.5 mM; MgSO₄ 0.8 mM; KH₂PO₄ 1.2 m; NaHCO₃ 25.0 mM; Glucose11.1 mM.

Formulations of the present invention also further comprise any one ormore of the following:

a steroidal anti-inflammatory drug (corticosteroid), a calcineurininhibitor (eg pimecrolimus, tacrolimus), a phosphodiesterase inhibitors,a anti-histamine, a anti-microbial agent, a antibiotic, a antibacterialagent, a ceremide, a growth factor (eg transforming growth factors β1-3,platelet derived growth factor, fibroblast growth factor, insulin-likegrowth factors I & II, epidermal growth factor, keratinocyte growthfactor, nerve growth factor), a mitogenic agent, a matrixmetalloproteinase inhibitor (eg TIMP's, Batimastat, Marimastat, andmatlystatin B), a protease inhibitor, a ECM protein, tretinoin (VitaminA), a antioxidant (vitamins E and C), a plant cytokinin (kinerase), acopper-peptide complexes as well as numerous plant, animal and mineralextracts (ie coal tar extract).

Specific formulations of the present invention comprise a combinationwith a steroidal anti-inflammatory drug. Another composition comprises acalcineurin inhibitor. Another composition comprises an anti-histamine.Another composition comprises an anti-microbial agent. Anothercomposition comprises a growth factor. Another composition comprises aprotease inhibitor.

Preparation of Compositions for Administration

Liposomes

Encapsulation of proteins within liposomes are detailed for example inU.S. Pat. Nos. 5,662,931; 5,853,755; 4,485,054; 5,780,054; 5,653,974;6,019,999; 6,027,726; 5,739,273; 5,264,221; 5,413,804; 5,374,715.

Polymers

The polysaccharides and/or polypeptides of the present invention, can beencapsulated within biodegradable synthetic polymers (or derivatives)for controlled release. These polymers (and derivatives) include forexample: Poly(esters); examples are poly(ε-caprolactone) PCL,poly(glycolic acid) PGA, poly(L-lactic acid) PLA, poly(ethylene glycol)PEG, poly(ethylene oxide) PEO. Poly(ester) derivatives includePoly(ester) copolymers, Poly(ortho esters). Poly(ester) copolymers;examples are poly(lactic acid-co-glycolic acid) PLGA, poly(D-lacticacid) PDLA, poly(L-lactic acid) PLLA, PLA-PEG, diblock PLA/PEG, triblockPLA/PEG/PLA. Poly(ortho esters); examples are3,9-diethylidene-2,4,8,10-tetraoxaspiro[5.5]undecane (DETOSU)-basedpoly(orthoesters). Poly(anhydrides); examples are sebacic acid (SA),p-(carboxyphenoxy)propane (CPP), p-(carboxyphenoxy)hexane (CPH), SA/CPPcopolymers, poly(fatty acid dimer-sebacic acid), poly(anhydride-imides),poly(anhydride-esters). Poly(amides); examples are poly(amino acids),poly(glutamic acid), poly(aspartic acid), poly(lactic acid-co-lysine)PLAL, poly[N-(3-hydroxypropyl)-L-glutamine], poly(iminocarbonates),tyrosine-derived poly(carbonates). Phosphorus-containing polymers; iepoly(phosphazenes), poly(dichlorophosphazenes),poly(organophosphazenes), poly[bis(carboxylatophenoxy)-phosphazene],poly(phosphoesters), poly(urethanes), a hyaluronan carrier.

Coupling

Polypeptide(s) and/or polysaccharide(s) can be coupled to a collagenmatrix for administration. Collagen matrix can release a coupled drug ata constant effective concentration. Accordingly, collagen and other ECMprotein matrices can effectively be used to administer polypeptide(s)and/or polysaccharide(s) in vivo, for example into a tissue or space inneed thereof. In one embodiment the cross-linked collagen matrix isadministered subcutaneously.

General

Generally, the present invention relates to and/or uses therapeuticallyeffective amounts and/or prophylactically effective amounts of thecompositions discussed herein.

A “therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredeffect.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result, such as preventing or inhibiting cell apoptosis ortissue damage.

Polypeptides

Reference herein to “polypeptide” includes single polypeptides, mixturesof polypeptides and also biologically active fragments of polypeptides.

By “substantially purified polypeptide” we mean a polypeptide that hasbeen at least partially separated from the lipids, nucleic acids, otherpolypeptides, and other contaminating molecules with which it isassociated in its native state. In one embodiment, the substantiallypurified polypeptide is at least 60% free from other components withwhich they are naturally associated. In a further embodiment, thesubstantially purified polypeptide is at least 75% free from othercomponents with which they are naturally associated. In a furtherembodiment, the substantially purified polypeptide is at least 90% freefrom other components with which they are naturally associated.Furthermore, the term “polypeptide” is used interchangeably herein withthe term “protein”.

The % identity of a polypeptide is determined by GAP (Needleman andWunsch, 1970) analysis (GCG program) with a gap creation penalty=5, anda gap extension penalty=0.3. Unless stated otherwise, the query sequenceis at least 15 amino acids in length, and the GAP analysis aligns thetwo sequences over a region of at least 15 amino acids. The querysequence may be at least 50 amino acids in length, and the GAP analysisaligns the two sequences over a region of at least 50 amino acids. Thequery sequence may at least 100 amino acids in length and the GAPanalysis aligns the two sequences over a region of at least 100 aminoacids.

With regard to the defined polypeptides/enzymes, it will be appreciatedthat % identity figures higher than those provided above will encompassembodiments. Thus, where applicable, in light of the minimum % identityfigures, the polypeptide may comprises an amino acid sequence which isat least 60%, 65%, 70%, 75%, 76%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, or 99.9% identical to the relevant nominated SEQ ID NO.

As used herein, the term “biologically active fragment” refers to aportion of the defined polypeptide which still maintains anti-arthriticor anti-inflammatory activity (whichever is relevant). Such biologicallyactive fragments can readily be determined by serial deletions of thefull length protein, and testing the activity of the resulting fragment.

Amino acid sequence mutants/variants of the polypeptides/enzymes definedherein can be prepared by introducing appropriate nucleotide changesinto a nucleic acid encoding the polypeptide, or by in vitro synthesisof the desired polypeptide. Such mutants include, for example,deletions, insertions or substitutions of residues within the amino acidsequence. A combination of deletion, insertion and substitution can bemade to arrive at the final construct, provided that the final proteinproduct possesses the desired characteristics.

In designing amino acid sequence mutants, the location of the mutationsite and the nature of the mutation will depend on characteristic(s) tobe modified. The sites for mutation can be modified individually or inseries, e.g., by (1) substituting first with conservative amino acidchoices and then with more radical selections depending upon the resultsachieved, (2) deleting the target residue, or (3) inserting otherresidues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 30residues, about 1 to 10 residues and typically about 1 to 5 contiguousresidues.

Substitution mutants have at least one amino acid residue in thepolypeptide molecule removed and a different residue inserted in itsplace. The sites of greatest interest for substitutional mutagenesisinclude sites identified as the active or binding site(s). Other sitesof interest are those in which particular residues obtained from variousstrains or species are identical. These positions may be important forbiological activity. These sites, especially those falling within asequence of at least three other identically conserved sites, may besubstituted in a relatively conservative manner. Such conservativesubstitutions are shown in Table A.

Furthermore, if desired, unnatural amino acids or chemical amino acidanalogues can be introduced as a substitution or addition into thepolypeptides of the present invention. Such amino acids include, but arenot limited to, the D-isomers of the common amino acids,2,4-diaminobutyric acid, D-amino isobutyric acid, 4-aminobutyric acid,2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,D-alanine, fluoro-amino acids, designer amino acids such as D-methylamino acids, C-methyl amino acids, N-methyl amino acids, and amino acidanalogues in general.

TABLE A Exemplary substitutions. Original Exemplary ResidueSubstitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; hisAsp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, alaHis (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; pheLys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S)thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe,ala

Also included within the scope of the invention are polypeptides of thepresent invention which are differentially modified during or aftersynthesis, e.g., by biotinylation, benzylation, glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. These modifications may serve toincrease the stability and/or bioactivity of the polypeptide of theinvention.

Polypeptides of the present invention can be produced in a variety ofways, including production and recovery of natural proteins, productionand recovery of recombinant proteins, and chemical synthesis of theproteins. In one embodiment, an isolated polypeptide of the presentinvention is produced by culturing a cell capable of expressing thepolypeptide under conditions effective to produce the polypeptide, andrecovering the polypeptide. One cell to culture is a recombinant cell ofthe present invention. Effective culture conditions include, but are notlimited to, effective media, bioreactor, temperature, pH and oxygenconditions that permit protein production. An effective medium refers toany medium in which a cell is cultured to produce a polypeptide of thepresent invention. Such medium typically comprises an aqueous mediumhaving assimilable carbon, nitrogen and phosphate sources, andappropriate salts, minerals, metals and other nutrients, such asvitamins. Cells of the present invention can be cultured in conventionalfermentation bioreactors, shake flasks, test tubes, microtiter dishes,and petri plates. Culturing can be carried out at a temperature, pH andoxygen content appropriate for a recombinant cell. Such culturingconditions are within the expertise of one of ordinary skill in the art.

Gene Therapy

The polynucleotides and polypeptides may be employed in accordance withthe present invention by expression of such polypeptides in treatmentmodalities often referred to as “gene therapy”. Thus, for example, cellsfrom a patient may be engineered with a polynucleotide, such as a DNA orRNA, to encode a polypeptide ex vivo. The engineered cells can then beprovided to a patient to be treated with the polypeptide. In thisembodiment, cells may be engineered ex vivo, for example, by the use ofa retroviral plasmid vector containing RNA encoding a polypeptide usefulfor the methods of the present invention to transform said cells. Suchmethods are well-known in the art and their use in the present inventionwill be apparent from the teachings herein.

Further, cells may be engineered in vivo for expression of a polypeptidein vivo by procedures known in the art. For example, a polynucleotideuseful for a method of the present invention may be engineered forexpression in a replication defective retroviral vector or adenoviralvector or other vector (for example, poxvirus vectors). The expressionconstruct may then be isolated. A packaging cell is transduced with aplasmid vector containing RNA encoding a polypeptide useful for a methodof the present invention, such that the packaging cell now producesinfectious viral particles containing the gene of interest. Theseproducer cells may be administered to a patient for engineering cells invivo and expression of the polypeptide in vivo. These and other methodsfor administering a polypeptide of the present invention should beapparent to those skilled in the art from the teachings of the presentinvention.

Retroviruses from which the retroviral plasmid vectorshereinabove-mentioned may be derived include, but are not limited to,Moloney Murine Leukemia Virus, Spleen Necrosis Virus, Rous SarcomaVirus, Harvey Sarcoma Virus, Avian Leukosis Virus, Gibbon Ape LeukemiaVirus, Human Immunodeficiency Virus, Adenovirus, MyeloproliferativeSarcoma Virus, and Mammary Tumor Virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

Such vectors will include one or more promoters for expressing thepolypeptide. Suitable promoters which may be employed include, but arenot limited to, the retroviral LTR; the SV40 promoter; and the humancytomegalovirus (CMV) promoter. Cellular promoters such as eukaryoticcellular promoters including, but not limited to, the histone, RNApolymerase III, and β-actin promoters, can also be used. Additionalviral promoters which may be employed include, but are not limited to,adenovirus promoters, thymidine kinase (TK) promoters, and B19parvovirus promoters. The selection of a suitable promoter will beapparent to those skilled in the art from the teachings containedherein.

The nucleic acid sequence encoding the polypeptide useful for a methodof the present invention will be placed under the control of a suitablepromoter. Suitable promoters which may be employed include, but are notlimited to, adenoviral promoters, such as the adenoviral major latepromoter; or heterologous promoters, such as the cytomegalovirus (CMV)promoter; the respiratory syncytial virus (RSV) promoter; induciblepromoters, such as the MMT promoter, the metallothionein promoter; heatshock promoters; the albumin promoter; the ApoAI promoter; human globinpromoters; viral thymidine kinase promoters, such as the Herpes Simplexthymidine kinase promoter; retroviral LTRs (including the modifiedretroviral LTRs herein above described); the β-actin promoter; and humangrowth hormone promoters. The promoter may also be the native promoterwhich controls the gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, Y-2,Y-AM, PA12, T19-14X, VT-19-17-H2, YCRE, YCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller (1990) Human Gene Therapy, 1:5-14.

The vector may be transduced into the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

The producer cell line will generate infectious retroviral vectorparticles, which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles may then be employed totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, mesenchemymal cells, chondrocytes, embryonic stem cells,embryonic carcinoma cells, as well as hematopoietic stem cells,hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells,and bronchial epithelial cells.

Genetic therapies in accordance with the present invention may involve atransient (temporary) presence of the gene therapy polynucleotide in thepatient or the permanent introduction of a polynucleotide into thepatient.

Genetic therapies, like the direct administration of agents discussedabove, in accordance with the present invention may be used alone or inconjunction with other therapeutic modalities.

Preparation and Administration of Pharmaceutical Compositions

The amount of polysaccharide optionally with a polypeptide to beadministered may vary according to factors such as the disease state,age, sex, and weight of the individual. Dosage regimens may be adjustedto provide the optimum therapeutic response. For example, a single bolusmay be administered, several divided doses may be administered over timeor the dose may be proportionally reduced or increased as indicated bythe exigencies of the therapeutic situation. It may be advantageous toformulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. “Dosage unit form” as usedherein refers to physically discrete units suited as unitary dosages forsubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

It will be appreciated that the polysaccharide optionally with apolypeptide may be administered in the form of a composition comprisinga pharmaceutically acceptable carrier or excipient.

As used herein “pharmaceutically acceptable carrier” or “excipient”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike that are physiologically compatible. In one embodiment, the carrieris suitable for parenteral administration. Alternatively, the carriercan be suitable for intravenous, intraperitoneal, intramuscular,sublingual or oral administration. Pharmaceutically acceptable carriersinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe pharmaceutical compositions of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, liposome, or other orderedstructure. The carrier can be a solvent or dispersion medium containing,for example, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol, orsodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, monostearatesalts and gelatin. Moreover, the polysaccharide and/or polypeptide maybe administered in a time release formulation, for example in acomposition which includes a slow release polymer. The active compoundscan be prepared with carriers that will protect the compound againstrapid release, such as a controlled release formulation, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylacticacid and polylactic, polyglycolic copolymers (PLG). Many methods for thepreparation of such formulations are patented or generally known tothose skilled in the art.

The polysaccharide optionally with a polypeptide may be administered incombination with an appropriate matrix, for instance, for providing asurface for bone, cartilage, muscle, nerve, epidermis and/or otherconnective tissue growth. The matrix may be in the form of traditionalmatrix biomaterials. The matrix may provide slow release of the cells,supernatant or soluble factors.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. Potential matrices for the compositions may bebiodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid and polyanhydrides. Otherpotential materials are biodegradable and biologically well defined,such as bone or dermal collagen. Further matrices are comprised of pureproteins or extracellular matrix components. Other potential matricesare nonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalcium phosphate. The bioceramics may be altered in composition,such as in calcium-aluminate-phosphate and processing to alter poresize, particle size, particle shape, and biodegradability.

Compositions of the invention may be prepared from one or morepolysaccharide and/or polypeptide. Additional polysaccharide and/orpolypeptide fragments or peptides can be identified by routineexperimentation in light of the present specification, claims andfigures. A method for identifying peptide fragments having stimulatoryactivity is described, for example, in U.S. Pat. No. 5,399,342.

The pharmaceutical compositions may be for human or animal usage inhuman and veterinary medicine and will typically comprise any one ormore of a pharmaceutically acceptable diluent, carrier or excipient.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described for example in Remington'sPharmaceutical Sciences Mack Publishing Co. (A. R. Gennaro edit. 1985).The choice of pharmaceutical carrier excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as, or in addition to, the carrier, excipient or diluent, anysuitable binder, lubricant, suspending agent, coating agent, orsolubilising agent.

It is well known in the art that there may be differentcomposition/formulation requirements dependant on the different deliverysystems.

According to the present invention non-invasive formulations are alsoencompassed. For example, while the progenitor cells are likely to beadministered parerentally, eg intra-articularly, the polysulfatedpolysaccharide can be administered by inhalation, orally orintranasally, in the form of suppository or pessary, topically in theform of a lotion, solution, cream, ointment, or dusting powder, by useof a skin patch, orally in the form of tablets containing excipientssuch as starch or lactose, or in capsules, chewables or ovules eitheralone or in admixture with excipients, or in the form of elixirs,solutions, syrups or suspensions containing flavouring or colouringagents.

For buccal or sublingual administrations, the compositions may beadministered for example in the form of tablets or lozenges which can beformulated in a conventional manner.

Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, and thelike. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders andcontain 10% to 95% of active ingredient, particularly 25% to 70%.Capsules, tablets and pills for oral administration to a patient may beprovided with an enteric coating comprising, for example, Eudragit “S”,Eudragit “L”, cellulose acetate, cellulose acetate phthalate orhydroxypropylmethyl cellulose.

Intranasal formulations are described and administration of larthryticcollagen type II and larthrytic collagen type IX are described forexample in Lu et al (1999) Different therapeutic and bystander effectsby intranasal administration of homologous type II and type IX collagenson the collagen-induced arthritis and pristane-induced arthritis inrats, Clinical Immunology Vol 90 pp 119-127 (1999).

In another example, the pharmaceutical composition of the presentinvention may be formulated to be delivered using a mini-pump or by amucosal route, for example, as a nasal spray or aerosol for inhalationor ingestible solution.

Where the agent is to be delivered mucosally through thegastro-intestinal mucosa, it should be able to remain stable duringtransit through the gastro-intestinal tract; for example, it should beresistant to proteolytic degradation, stable antacid, pH and resistantto the detergent effects of bile.

In one embodiment, the polysulfated polysaccharides of the invention areadministered by a non-invasive route. In a further embodiment, thenon-invasive route comprises oral administration, or enteraladministration, nasal administration or by inhalation.

In an alternate embodiment, compositions of the invention can beinjected parenterally for example, intravenously, intramuscularly orsubcutaneously.

For parenteral administration, the compositions may be best used in theform of a sterile aqueous solution which may contain other substances,for example enough salts or monosaccharides to make the solutionisotonic with blood. The preparation may also be emulsified, orencapsulated in liposomes.

After formulation, the immuno-protective composition may be incorporatedinto a sterile container which is then sealed and stored at a lowtemperature, for example 4° C., or it may be freeze-dried.Lyophilisation permits long-term storage in a stabilised form.

In one embodiment of the present invention progenitor, particular thechondroprogenitor and even more particularly the Stro-1^(bri) cellsand/or progeny cells thereof are administered in the form of acomposition. In one embodiment, such a composition comprises apharmaceutically acceptable carrier and/or excipient.

The terms “carrier” and “excipient” refer to compositions of matter thatare conventionally used in the art to facilitate the storage,administration, and/or the biological activity of an active compound(see, e.g., Remington's Pharmaceutical Sciences, 16th Ed., MacPublishing Company (1980). A carrier may also reduce any undesirableside effects of the active compound. A suitable carrier is, for example,stable, e.g., incapable of reacting with other ingredients in thecarrier. In one example, the carrier does not produce significant localor systemic adverse effect in recipients at the dosages andconcentrations employed for treatment.

Suitable carriers for this invention include those conventionally used,e.g., water, saline, aqueous dextrose, lactose, Ringer's solution, abuffered solution, hyaluronan and glycols are preferred liquid carriers,particularly (when isotonic) for solutions. Suitable pharmaceuticalcarriers and excipients include starch, cellulose, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesiumstearate, sodium stearate, glycerol monostearate, sodium chloride,glycerol, propylene glycol, water, ethanol, and the like.

In another example, a carrier is a media composition, e.g., in which acell is grown or suspended. In a further example, such a mediacomposition does not induce any adverse effects in a subject to whom itis administered.

Preferred carriers and excipients do not adversely affect the viabilityof a cell.

In one example, the carrier or excipient provides a buffering activityto maintain the cells and/or soluble factors at a suitable pH to therebyexert a biological activity, e.g., the carrier or excipient is phosphatebuffered saline (PBS). PBS represents an attractive carrier or excipientbecause it interacts with cells and factors minimally and permits rapidrelease of the cells and factors, in such a case, the composition of theinvention may be produced as a liquid for direct application to theblood stream or into a tissue or a region surrounding or adjacent to atissue, e.g., by injection.

Progenitor, particular the chondroprogenitor and even more particularlyStro-1^(bri) cells and/or progeny cells thereof can also be incorporatedor embedded within scaffolds that are recipient-compatible and whichdegrade into products that are not harmful to the recipient. Thesescaffolds provide support and protection for cells that are to betransplanted into the recipient subjects. Natural and/or syntheticbiodegradable scaffolds are examples of such scaffolds.

A variety of different scaffolds may be used successfully in thepractice of the invention. Scaffolds include, but are not limited tobiological, degradable scaffolds. Natural biodegradable scaffoldsinclude collagen, fibronectin, and laminin scaffolds. Suitable syntheticmaterial for a cell transplantation scaffold should be able to supportextensive cell growth and cell function. Such scaffolds may also beresorbable. Suitable scaffolds include polyglycolic acid scaffolds,e.g., as described by Vacanti, et al. J. Ped. Surg. 23:3-9 1988; Cima,et al. Biotechnol. Bioeng. 38:145 1991; Vacanti, et al. Plast. Reconstr.Surg. 88:753-9 1991; or synthetic polymers such as polyanhydrides,polyorthoesters, and polylactic acid.

In another example, the cells may be administered in a gel scaffold(such as Gelfoam from Upjohn Company).

The cellular compositions useful for the present invention may beadministered alone or as admixtures with other cells. Cells that may beadministered in conjunction with the compositions of the presentinvention include, but are not limited to, other multipotent orpluripotent cells or stem cells, or bone marrow cells. The cells ofdifferent types may be admixed with a composition of the inventionimmediately or shortly prior to administration, or they may beco-cultured together for a period of time prior to administration.

In one embodiment, the composition comprises an effective amount or atherapeutically or prophylactically effective amount of cells. Forexample, the composition comprises about 1×10⁵ Stro-1^(bri) cells/kg toabout 1×10⁷ Stro-1^(bri) cells/kg or about 1×10⁶ Stro-1^(bri) cells/kgto about 5×10⁶ Stro-1^(bri) cells/kg. The exact amount of cells to beadministered is dependent upon a variety of factors, including the age,weight, and sex of the patient, and the extent and severity of thepancreatic dysfunction. The same values are also applicable to theprogenitor cells and chondroprogenitor cells per se.

In some embodiments, cells are contained within a chamber that does notpermit the cells to exit into a subject's circulation, however thatpermits factors secreted by the cells to enter the circulation. In thismanner soluble factors may be administered to a subject by permittingthe cells to secrete the factors into the subject's circulation. Such achamber may equally be implanted at a site in a subject to increaselocal levels of the soluble factors, e.g., implanted in or near atransplanted organ.

In some embodiments of the invention, it may not be necessary ordesirable to immunosuppress a patient prior to initiation of therapywith cellular compositions. Accordingly, transplantation withallogeneic, or even xenogeneic, Stro-1^(bri) cells or progeny thereofmay be tolerated in some instances.

However, in other instances it may be desirable or appropriate topharmacologically immunosuppress a patient prior to initiating celltherapy. This may be accomplished through the use of systemic or localimmunosuppressive agents, or it may be accomplished by delivering thecells in an encapsulated device. The cells may be encapsulated in acapsule that is permeable to nutrients and oxygen required by the celland therapeutic factors the cell is yet impermeable to immune humoralfactors and cells. The encapsulant may be hypoallergenic, is easily andstably situated in a target tissue, and provides added protection to theimplanted structure. These and other means for reducing or eliminatingan immune response to the transplanted cells are known in the art. As analternative, the cells may be genetically modified to reduce theirimmunogenicity.

Uses and Methods of Treatment

The methods, compositions, and uses of the present invention are usefulfor the treatment and/or prophylaxis of diseases of the musculoskeletalsystem such as rheumatoid arthritis (RA), osteoarthritis (OA) andintervertebral disc degeneration (DD). They can also be usefullyemployed in relation to cartilage regeneration and repair.

The methods, compositions, and uses of the present invention are alsouseful in that they can regulate chondrogenesis and cell proliferationand can be used to produce, upregulate or stimulate the production ofhyaluronan (HA). These uses can be employed in the treatment of diseasesof the musculoskeletal system including rheumatoid arthritis (RA),osteoarthritis (OA), and intervertebral disc degeneration (DD); ortreating conditions that benefit from increased production of HA, suchas for example osteoarthritis of synovial joints, ophthalmology,prevention of post-surgical abdominal adherences, skin treatment andrepair and restoration of the function of the extracellular matrix; orinducing cartilage repair, restoration or matrix neogenesis.

Further uses of the present invention include producing an extracellularmatrix suitable for transplantation into a connective tissue defect in asubject in need of such a treatment.

The methods of the present invention, can therefore be used to treat apatient, in some embodiments a human patient, from a number of diseasesas stated above. The methods can also be used on a prophylactic basis toprevent or minimise the onset of these diseases.

The present invention also extends to compositions as discussed hereinfor use in the treatment and/or prophylaxis of diseases of themusculoskeletal system such as rheumatoid arthritis (RA), osteoarthritis(OA) and intervertebral disc degeneration (DD). They can also beusefully employed in relation to cartilage regeneration and repair, ortreating conditions that benefit from increased production of HA, suchas for example osteoarthritis of synovial joints, ophthalmology,prevention of post-surgical abdominal adherences, skin treatment andrepair and restoration of the function of the extracellular matrix; orinducing cartilage repair, restoration or matrix neogenesis.

The compositions as discussed herein can also be used in producing anextracellular matrix suitable for transplantation into a connectivetissue defect in a subject in need of such a treatment and also forregulating chondrogenesis and cell proliferation and/or producing,upregulating or stimulating the production of hyaluronan (HA).

The present invention also extends to the use of compositions asdiscussed herein in the manufacture of a medicament for the treatmentand/or prophylaxis of diseases of the musculoskeletal system such asrheumatoid arthritis (RA), osteoarthritis (OA) and intervertebral discdegeneration (DD). The use also extends to cartilage regeneration andrepair, or treating conditions that benefit from increased production ofHA, such as for example osteoarthritis of synovial joints,ophthalmology, prevention of post-surgical abdominal adherences, skintreatment and repair and restoration of the function of theextracellular matrix; or inducing cartilage repair, restoration ormatrix neogenesis.

The use of compositions as discussed herein also extends to themanufacture of a medicament for producing an extracellular matrixsuitable for transplantation into a connective tissue defect in asubject in need of such a treatment and for regulating chondrogenesisand cell proliferation and/or producing, upregulating or stimulating theproduction of hyaluronan (HA).

The present invention allows for the administration of the compositionsof the present invention to implant progenitor cells into a patientwhich are subsequently induced to increase HA production and/or undergotransformation into a chondrogenic phenotype.

Examples of such an application would be to inject the compositions ofthe present invention into joints of individuals with cartilage or disclesions or systemically for other less accessible sites, allowing thepreparation to perfuse the tissue and cells thereby exerting its uniquebiological effects. Applications could include any treating individualswho may not have clinical defined disease (often OA or relateddisorders) but have sustained a traumatic injury to joint tissues thoughsport or work-related activity.

In older subjects with OA or related disorders this form of treatmentcould be used instead of intra-articular HA therapy(viscosupplementation).

It could also serve as a prophylactic method following arthroscopic oropen surgery where cartilage or meniscal excision/debridement wasnecessary. It is well established that with time such post surgicalpatients will generally progress to exhibit symptomatic OA requiringmedical treatment. It is not unlikely that by diminishing cartilagedegradation symptoms may also improved because of the reduction inproduction of cartilage derived auto-antigens which promoteinflammation.

Compositions according to the invention that have been shown to haveactivity can be further tested for safety and efficacy in other animalmodels, and then proceed to clinical trials in humans, if desired.Naturally, for veterinary applications, no clinical trial in humans isrequired. Those compositions that are safe and efficacious in animals orhumans can be administered to an appropriate subject to treat oralternatively to protect against the diseases discussed herein.“Treatment and protection” includes both prophylactic and therapeuticmeasures to prevent the onset and appearance of diseases as discussedherein.

The treatment methods herein refers to defending against or inhibiting asymptom, delaying the appearance of a symptom, reducing the severity ofthe development of a symptom, and/or reducing the number or type ofsymptoms suffered by an individual, as compared to not administering apharmaceutical composition comprising a polypeptide of the invention.Accordingly, throughout this description, it will be understood that anyclinically or statistically significant attenuation of even one symptomof a musculoskeletal degenerative condition pursuant to the treatmentaccording to the present invention is within the scope of the invention.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

Results and Discussion

The present invention has shown that the addition of polysulfatedpolysaccharides over a wide range of concentrations to cryogenic mediasuch as Profreeze® and 7.5% DMSO containing progenitor cells in bothhigh and low numbers followed by freezing in liquid nitrogen vapourphase and thawing at ambient temperatures or 37° C. had no detrimentaleffect on their viability. This can be seen by FIGS. 1-3. Indeed,enhanced viability was seen from these experiments, in particular FIG. 3and also FIG. 4 which shows that progenitor cell viability was enhancedrelative to progenitor cells frozen in cryo-preservation media notcontaining the polysulfated polysaccharide.

FIG. 6 clearly shows that polysulfated polysaccharide concentrationsabove 1 microgram/mL reduced apoptosis in human progenitor cells byabout 50% when these cells were incubated with IL-4 and IFN-gamma whichwere known mediators of apoptosis.

It has also been shown that polysulfated polysaccharides stimulateprogenitor cell proliferation in a concentration dependent manner.Marked stimulation of cell division, as measured with the WST-1mitchondral dehydrogenase cleavage assay is seen in FIG. 4 and fromincorporation of ³H-Thymidine into DNA in FIG. 5 which was demonstratedover the range of 1-5 micrograms/mL in monolayer and micromass culturesof human progenitor cells as well as in a murine progenitor cell line inFIG. 10.

In contrast to several members of the BMP-TGF-beta super family (eg,BMP-2, BMP-7, BMP-8) and fibroblast growth factor family which promotedifferentiation of progenitor cells to osteoblasts when cultured inosteogenic media, it was found for the first time that polysulfatedpolysaccharides suppress differentiation of progenitor cells to thiscell phenotype. This can be seen in FIG. 7 and shown downregulation ofthe progenitor cell with regard to osteogenesis.

On the other hand progenitor cells cultured in adipogenic media in thepresence polysulfated polysaccharides showed differentiated toadipocytes. Thus, polysulfated polysaccharides act as a regulator ofprogenitor cell differentiation into adipocytes. This can be seen inFIG. 8.

Under normal non-selective culture conditions incubation of polysulfatedpolysaccharides with progenitor cells invariably favoureddifferentiation along the chondrogenic pathway as demonstrated byincreased proteoglycan and type II collagen synthesis. Proteoglycans andtype II collagen are recognised biosynthetic products of chondrocytesand are used as phenotypic markers of hyaline cartilage. Thechondrogenic promoting effect of polysulfated polysaccharides was shownin the Murine MSC cell line C3H10T1/2 in FIG. 9 and human progenitorcells in FIG. 11 when cultured in monolayers.

Additional support was provided in pellet cultures using the MurineATDC5 cell line where a 25% increase of proteoglycan (PG) synthesisrelative to control culture (no polysulfated polysaccharides) wasobserved at 2 micrograms/mL in FIG. 12.

Additional evidence that polysulfated polysaccharides promotedchondrogenesis and cartilage formation was provided by examination ofgene expression by these cells after 6 days in pellet culture, wheretype II collagen expression, was up-regulated in a concentrationdependent manner by polysulfated polysaccharides. This can be seen inFIG. 13.

Interestingly, Heparin, a naturally occurring polysulfatedpolysaccharide failed to significantly stimulate proteoglycan synthesisin pellet culture over all of the concentrations examined. This can beseen in FIG. 14. Heparin shows little activity at the concentration of2.5 ugrams/mL but may cause inhibition at higher concentrations.

Using the Murine MSC line C3H10T1/2 in 7 day pellet culture demonstratedthat polysulfated polysaccharides at 10 micrograms/mL increasedproteoglycan synthesis by more than 80% of the control values. This canbe seen in FIG. 15. This finding was consistent with the resultsobtained using a Murine MSC line C3H10T1/2 in micromass cultures over 6days which can be seen in FIG. 16. This method of culturing progenitorcells was originally described by Denker et al (Andrew E. Denker A E,Haas A R, Nicoll S B, Tuan R S. Chondrogenic differentiation of murineC3H10T1/2 multipotential progenitor cells: I. Stimulation by bonemorphogenetic protein-2 in high-density micromass cultures.Differentiation (1999) 64:67-76 1999).

Moreover, after nine days in micromass cultures a 100% stimulation wasobtained at 1 microgram/mL of polysulfated polysaccharides also shown inFIG. 16. Human progenitor also differentiated to chondrocytes inmicromass cultures when incubated in the presence of polysulfatedpolysaccharides. However in the 5 day cultures a maximum stimulation ofPG synthesis of 30% was obtained with polysulfated polysaccharidesconcentration of 2.5 micrograms/mL as shown in FIG. 17. Theco-production of type II collagen with PGs was confirmed in thesemicromass cultures using the immuno-staining technique described byDenker et al (Andrew E. Denker A E, Haas A R, Nicoll S B, Tuan R S.Chondrogenic differentiation of murine C3H10T1/2 multipotentialprogenitor cells: I. Stimulation by bone morphogenetic protein-2 inhigh-density micromass cultures. Differentiation (1999) 64:67-76 1999).

As is evident from FIG. 18, micromass cultures of human progenitor cellsmaintained in the presence of polysulfated polysaccharides over theconcentration range of 1-10 micrograms/mL for 10 days afforded intensetype II collagen staining, the maximum levels being achieved withpolysulfated polysaccharides at 5 micrograms/mL.

In similar micromass cultures undertaken with human progenitor cells andhyaluronan (HA) a low level of ³⁵SO4 incorporation into PGs was observedin FIG. 19A. However, the highly negatively charged Dextran Sulfate (DS)inhibited PG synthesis over the range of 1-20 micrograms/mL (FIG. 19B).This was a surprising result since DS has a similar molecular weight andcharge density to polysulfated polysaccharides and was thereforeexpected to demonstrate similar activity on the progenitor cells. It isbelieved that molecular conformation and other factors important foreffective receptor binding and protein interactions may be playing arole.

Hyaluronan is reported to exert chondrogenic effects on progenitor cellsin alginate bead cultures (Kavalkovich K W, Boynton R E, Murphy J M,Barry F. Chondrogenic differentiation of human progenitor cells withinan alginate layer culture system. In vitro Cell. Dev. Biol—Animal.38:457-466, 2002). In FIG. 20 the effects of Pentosan Polysulfate aloneand in combination with Hyaluronan (Supartz™) on human progenitor cellproliferation using the WST-1 assay is shown for day 3 and day 5cultures. The results of this experiment confirmed the absence of anysignificant stimulatory effect by HA alone on progenitor cellproliferation and also demonstrate the absence of any synergist effectfor the combinations with polysulfated polysaccharides. Thus, it can beseen that polysulfated polysaccharides are better chondrogenic agentsthan HA. In addition, in contrast with NC4, HA does not combinesynergistically with polysulfated polysaccharides.

A recombinant human preparation of the non collagenous domain of thealpha-1 chain of type IX collagen, rhNC4 was also found to inducechondrogenesis and PG production by progenitor cells. As is evident fromFIG. 21, a concentration dependent stimulation of PG synthesis wasobserved both in the absence (maintenance media) and presence of Insulin(differentiation media) on ATDC5 cells, maximum effects occurring at 1microgram/mL. This stimulatory effect of rhNC4 was also demonstrated inpellet cultures of ATDC5 cells but in maintenance media the optimumeffect was produce at a concentration of 0.5 micrograms/mL.

The chondrogenic and mitogenic effects of rhNC4 on Murine ADTC5 cellswere observed to be enhanced when the protein was co-cultured withpolysulfated polysaccharides as can be seen in FIGS. 23 and 24. Thecombination of rhNC4 and polysulfated polysaccharides was, in contrastto HA and polysulfated polysaccharides, synergistic as shown by a 450%stimulation of ³H-DNA synthesis in differentiation culture media atconcentrations of 1 microgram/mL of rhNC4 and 2 micrograms/mL ofpolysulfated polysaccharides (FIG. 23). In terms of stimulation of PGsynthesis, 1-5 micrograms/mL rhNC4 with 5 micrograms polysulfatedpolysaccharides appeared to be the most effective combination (FIG. 24).

FIGS. 21, 33 and 23 show the outcome of experiments using both adifferentiation medium (DM) for the progenitor cells (ATDC5) containinga growth factor (in this case insulin) and the maintenance media (MM)which does not contain a growth factor. FIG. 21 shows the use of NC4alone, while FIGS. 33 and 23 show the effects of the combination of thepolypeptide and the polysulfated polysaccharide. It can be seen that thecompounds of the present invention promoted chondrogenesis in theabsence of the growth factor but also had a positive effect on the rateof chondrogenesis in the presence of the growth factors. Therefore, thecompounds of the present invention can be used without other growthfactors but can also be used with other growth factors to promotechondrogenesis further.

Studies of the expression of Runx 2 gene by ATDC5 cells cultured in thepresence of rhNC4 and polysulfated polysaccharides showed a progressivedown regulation of this bone marker with increasing concentrations(FIGS. 25 and 26). By contrast one of the genes for HA synthesis (HAS3)and the receptor for HA (CD44) were both up regulated by these compounds(FIGS. 25 and 26). Of particular interest was the finding of the upregulation of Smad 2 and Smad 4 by polysulfated polysaccharides aloneand polysulfated polysaccharides+rhNC4 at concentrations which wereshown to stimulate PG synthesis. Smad 4 has been reported to be a majorintracellular protein for the transactivation of the type-II collagenpromoter in progenitor cells when these cells are activated by BMPs(Hatakeyama Y et al. Smad signaling in progenitor and chondroprogenitorcells. J Bone Joint Surg AM. 85A Suppl 3, 13-8, 2003, Chen D, Zhao M,Munday G R, Bone morphogenetic proteins. Growth Factors, 22: 233-41,2004). Whilst not wishing to be bound by theory, it is possibletherefore that the chondrogenic/proliferatatory effects of polysulfatedpolysaccharides and NC4 on progenitor cells could be mediated via theactions of BMPs, the levels of which are elevated in the presence ofthese two compounds when used alone or in combination.

Experimental Methods and Protocols

Determination of Protein Content of Samples Using the Bicinchoninic Acid(BCA) Assay

The protein content of all samples was determined using BCA assay (SmithP K, Krohn R I, Hermanson G T, Mallia A K, Gartner F H, Provenzano M D,Fujimoto E K, Goeke N M, Olson B J and Klenk D C. Anal. Biochem. 150,76-85, 1985). Freeze dried protein samples were directly dissolved inH2O to provide a 2.0 mg/ml solution. 20 μl of each sample solution wasadded to a well of 96-well plates. Just prior to assay, 50 parts ofreagent 1 (0.4% NaOH; 1.7% Na₂CO₃; 0.95% NaHCO₃; 1.0% bicinchoninicacid; 0.16% Na₂-tartrate) was mixed with reagent 2 (4% CuSO₄.5H₂O). 200μl of working reagent was added to the sample solution. After incubationat 37° C. for 60 min the absorbance A562 was read using a Thermomaxmicroplate reader. Bovine serum albumin (BSA) or highly purifiedgelatine (Gibco) at 0-10 μg/well were used to construct a standardcurve. The protein content of samples were determined from this standardcurve.

Analysis of Proteins by SDS-PAGE Electrophoresis

The method used is based on that described by Laemmli (Laemmli UK.Clevage of structural protein during the assembly of the headbacteriophage T4. Nature. 1970; 227:680-685). Briefly samples were mixed1:1 with 2× sample loading buffer (0.07 M TrisHCl, 1.5% SDS, 20%glycerol, 0.2M DTT and 0.1% BPB) to achieve the final concentrations ofbetween 4.0-20 mg/ml. The mixture was boiled in a water bath for 5 min.20 μl of solution were loaded into the wells of 8-16% pre-castTris-glycine gel (Norvex). SeeBlue pre-stained low molecular weightrange protein markers (Norvex) were loaded into wells on the left-handside of the gel and electrophoresis was performed at 125 V for 2 h. Thegel was stained in Coomassie blue R250 solution (40% ethanol, 10% aceticacid and 0.2% Coomassie R250) for 30 min and destained in a solutioncontaining 10% ethanol and 7.5% acetic acid for 16 h. The gel was driedin a Bio-Rad Gelair drier.

Sulfated Glycosaminoglycan (S-GAG) Assay Using the DMMB Dye

The sulfated glycosaminoglycan (S-GAG) concentrations in samples weredetermined using a colorimetric dye binding assay modified from thatdescribed by Farndale et al. (Farndale R W, Buttle D J, Barrett A J.Improved quantitation and discrimination of sulphated glycosaminoglycansby use of dimethyl methylene blue. Biochem. et. Biophys. Acta. 1986;883:173-177).

The assay is based on a metachromatic shift in absorption maxima from690 nm to 535 nm when a complex is formed between a mixture of1,9-dimethylmethylene blue (DMMB) and the sulfated-GAG in the sample orin a standard solution. The dye solution was made by adding 16 mg of1,9-dimethylmethylene blue to 5 ml ethanol to 2 g of sodium formate and2 ml of formic acid in a total volume of 1 liter at pH 3.5.

Chondroitin-6-sulfate (CS-C) standards (0-15 and 0-40 μg/ml: 50 μl) orsamples (50 μl) were transferred to a microtitre plate. The dye solution(200 μl) was added immediately to each well and absorbance was measuredat 540 nm, immediately as a precipitate will form on standing. Astandard curve was plotted using the absorption of samples of known CS-Cconcentration and the plate reader software. The concentration of theS-GAG in the unknown samples were determined from the CS-C standardcurve.

Hyaluronan (HA) ELISA

Ninety six well microtiter plates (Maxisorp®, Nunc) were coated at 4° C.overnight with umbilical cord HA (Sigma Chemical Co) (100 μl/well)dissolved in the coating buffer. Uncoated areas were then blocked with150 μl/well of 1% (w/v) BSA in phosphate buffer saline (PBS) for 60 minat 25° C. After washed with PBS-Tween, 100 μl of the samples to beassayed or standard competitor (HA Healon®: range 19.53-10,000 ng/ml)together with Biotin conjugated-HA-binding protein (1:200) were added.After incubation for 60 min at 25° C., plates were washed and then aperoxidase-mouse monoclonal anti-biotin (Invitrogen) (100 μl/well;1:4,000) was added and the mixture incubated for 60 min at 25° C. Theplates were washed again and then a peroxidase substrate (Invitrogen)(100 μl/well) was added and incubated at 37° C. for 10-20 minutes toallow the color to develop. The reaction was stopped by addition of 50μl of 4 M H2SO4. The absorbance ratio at 492/690 nm was measured usingthe Titertek Multiskan M340 multiplate reader.

Semi-Quantitative RT-PCR mRNA

Total RNA was isolated from Progenitor cells according to themanufacturer's instructions for the Aurum total RNA mini kit (Bio-Rad,USA). The RNA was reverse transcribed with RevertAid™ H Minus FirstStand cDNA Synthesis Kit (Fermentas, USA). Table2 1 and 2 show theprimer sequences and condition used for PCR. PCR products weretransferred to an agarose gel, and visualized by ethidium bromidestaining, and integrated densities calculated using Scion image analysissoftware, normalized to the house-keeping geneGlyceraldehydes-3-phosphate dehydrogenase (GAPDH) to permitsemi-quantitative comparisons in mRNA levels. (L. Marchuk, P. Sciore, C.Reno, C. B. Frank, D. A. Hart. Postmortem stability of total RNAisolated from rabbit ligament, tendon and cartilage, Biochim BiophysActa. 1379 (1998) 171-177. R. Boykiw, P. Sciore, C. Reno, L. Marchuk, C.B. Frank, D. A. Hart. Altered levels of extracellular matrix moleculemRNA in healing rabbit ligaments, Matrix Biol. 17 (1998) 371-378.)

TABLE 1 Primers for Murine Reverse-Transcript PCR Gene Product (Murine)Primer Sequence Tm Cycles Size Protocol GADPHF: 5′CAC CAT GGA GAA GGC CGG GG 3′ 55 28 418 RT130308R: 5′GAC GGA CAC ATT GGG GGT AT 3′ SOX-9F: 5′CTG AAG GGC TAC GAC TGG AC 3′ 58 28 406 RT040308R: 5′GAG GAG GAA TGT GGG GAG TC 3′ AggrecanF: 5′AGG AGG TGG TAC TGC TGG TG 3′ 55 28 448 RT130308R: 5′TCT CAC TCC AGG GAA CTC GT 3′ Type IIF: 5′AGT CAA GGG AGA TCG TGG TG 3′ 58 28 598 RT040308 CollagenR: 5′CGT CGT GCT GTC TCA AGG TA 3′ ALPHF: 5′GCC CTC TCC AAG ACA TAT A 3′ 55 28 372 RT130308R: 5′CCA TGA TCA CGT CGA TAT CC 3′

TABLE 2 Primers used for semi-quantitative RT-PCR Product size Annealing(base Gene (HUMAN) temp (° C.) pairs) Sequences (5′ to 3′) Aggrecan 65110 Forward: ACTTCCGCTGGTCAGATGGA Reverse: TCTCGTGCCAGATCATCACCCollagen II 65 106 Forward: CAACACTGCCAACGTCCAGATReverse: CTGCTTCGTCCAGATAGGCAAT SOX9 68 101Forward: ACACACAGCTCACTCGACCTTG Reverse: GGAATTCTGGTTGGTCCTCTCTT hsp 7060 590 Forward:TTTGACAACAGGCTGGTGAACC Reverse:GTGAAGGATCTGCGTCTGCTTGGHAS 1 65 348 Forward:CGGCCTGTTCCCCTTCTTCGTGReverse: TCGTGTGCTACGCTGCGGACCA HAS2 51 358 Forward:CACAGCTGCTTATATTGTTGReverse: AGTGGCTGATTTGTCTCTGC HAS3 55 317 Forward:CAGCCTCCTCCAGCAGTTCCReverse: TAACCGTGGCAATGAGGAAG HYAL1 51 208Forward: AGCTGGGAAAATACAAGAACC Reward: TGAGCTGGATGGAGAAACTGG HYAL2 55448 Forward: GAGTTCGCAGCACAGCAGTTC Reward: CACCCCAGAGGATGACACCAG HYAL365 500 Forward: CCGCCTCCAGTGCCCTCTTCC Reward: AGCCCAGCCCCAGTAACAGTGMMP-1 68 84 Forward: CTGTTCAGGGACAGAATGTGCTReverse: TCGATATGCTTCACAGTTCTAGGG MMP-2 56 ~100Forward: TCAAGTTCCCCGGCGAT Reverse: TGTTCAGGTATTGCACTGCCA MMP-3 65 138Forward: TTTTGGCCATCTCTTCCTTCA Reverse: TGTGGATGCCTCTTGGGTATC MMP-9 56~100 Forward: TGAGAACCAATCTCACCGACAG Reverse: TGCCACCCGAGTGTAACCATMMP-13 65 96 Forward: TCCTCTTCTTGAGCTGGACTCATTReverse: CGCTCTGCAAACTGGAGGTC Human- 60 340Forward: CAGTACGTTTGGCAATGGAGACTGC iNOSReverse: GGTCACATTGGAGGTGTAGAGCTTG □5-Integrin 55 324Forward: CATTTCCGAGTCTGGGCCAA Reverse: TGGAGGCTTGAGCTGAGCTT □1-Integrin55 452 Forward: TGTTCAGTGCAGAGCCTTCA Reverse: CTTCATACTTCGGATTGACCFibronectin 56 143 Forward: CAT TCA CTG ATG TGG ATG TC Extra domain AReverse: CAG TGT CTT CTT CAC CAT CA Fibronectin  56 129Forward: CCG CCA TTA ATG AGA GTG AT Extra domain BReverse: AGT TAG TTG CGG CAG GAG AAG Total- 60 184Forward: GAT AAA TCA ACA GTG GGA GC fibronectinReverse: CCC AGA TCA TGG AGT CTT TA CD44 56 602Forward: GATCCACCCCAATTCCATCTGTGC Reverse: AACCGCGAGAATCAAAGCCAAGGCCADAMTS1 60.4 ~100 Forward: GAACAGGTGCAAGCTCATCTGReverse: TCTACAACCTTGGGCTGCAAA ADAMTS4 56 ~100Forward: CAAGGTCCCATGTGCAACGT Reverse: CATATGCCACCACCAGTGTCT ADAMTS560.4 ~100 Forward: TGTCCTGCCAGCGGATGT Reward: ACGGAATTACTGTACGGCCTACAGAPDH 53 370 Forward: TGGTATCGTGGAAGGACTCATReward: GTGGGTGTCGCTGTTGAAGTC

Separation of Peptacan Proteins/Polypeptides Using Dowex MAC3 CationExchange Resin

The Dowex MAC3 resin (100 grams) (Sigma Chemical Co) was regenerated asthe hydrogen form over 24 hours using 4% HCl. By means of a sinter-glassfilter the resin was thoroughly washed (3×2 L H2O) then equilibratedwith 0.1 M calcium acetate adjusted to pH 4.5. The Peptacan (10 grams)was dissolved in 0.1M calcium acetate, pH 4.5 at a concentration of 5mg/ml then mixed with the resin and gently agitated for 1 hr. Thesolution containing the non-bound S-GAGs and proteins was separated fromthe resin by filtration and the resin washed with loading buffer (10×resin volume) until no S-GAGs could be detected using the Farndale et alassay. The resin was further washed several time, with Milli-Q water andthen equilibrated with 0.2M Na2HPO4. The proteins bound to the resinwere released by a solution of 0.2M Na2HPO4 adjusted to pH 10.5 withNaOH. The resin was again separated by filtration through a sinter-glassBuchner funnel, the filtrate was collected and diafiltrated using a 1KDa TFF membrane (Millipore Australia Pty Ltd, Sydney, Australia) thenfreeze dried.

Separation of Peptacan Proteins/Polypeptides Using the CetylpyridiniumChloride (CPC) Precipitation Method

Cetyl pyridinium chloride (CPC) is a water soluble surfactant whichforms strong water insoluble complexes between its positively chargedpyridinium ion and the negatively charged sulfate groups present on thesulfated glycosaminoglycan (S-GAG) components of Peptacans.

These water insoluble CPC-S-GAG complexes have been extensively usedover the last 40 years by many investigators to isolate and purifyS-GAGs from extracts of biological tissues or fluids. However, wereasoned that this principle could also be used to isolate theproteins/polypeptides present in the Peptacans, since afterprecipitation and removal of the CPC-S-GAG complexes the Peptacanproteins would be left in the filtration liquors. The procedure used forthe CPC-S-GAG precipitation step was based on the method described byOegema and Thompson (Oegema T R and Thompson R C. Characterisation of ahyaluronic acid-dermatan sulfate proteoglycan complex fromdediffertiated human chondrocyte cultures. J Biol Chem. 256:1015-1022;1981) but modified by using 2M calcium chloride to dissociate theGAG-CPC complex and precipitating the S-GAGs from aqueous solution with4× the aqueous volume of ethanol. The aqueous solution containing thePeptacan proteins/polypeptides, after removal of the CPC-S-GAG complexwas extensively diafiltration using a 1000 Da cut-off ultrafiltrationmembrane (YC10) or a tangental flow ultrafiltration (TFF) cartridge ofsimilar MW cut-off (Millipore Australia Pty Ltd, Sydney, Australia).However, in the final stages of diafiltration the dialysing solution wasreplaced by 0.001 M acetic acid to avoid precipitation of the proteins.The diafiltrated solutions were freeze-dried to afford theprotein/polypeptides as a white powder.

Preparation of rhNC4 Using a Bacterial System.

The gene of NC4 of human collagen IX without the 23 signal peptide wasconstructed on the pGAT-2 bacterial expression vector in-frame with thesequences for the GST fusion tag based on a previously described method(Pihlajama T, et al. Characterization of Recombinant Amino-terminal NC4Domain of Human Collagen IX: Interaction with glycosaminoglycans andcartilage oligomeric matrix protein. J. Biol. Chem. 2004; 279:24265-24273). The recombinant human NC4 (rhNC4) construct wastransferred into a Escherichia coli BL21 (DE3) cell line. The fusionprotein was expressed in shaker flasks by inoculating (1:100) the cellline into the desired final volume of LB medium supplemented with 100microg/ml ampicillin. The cells were grown at 37° C. until absorbance at600 nm reached the value 0.6. The expression was induced by addition ofisopropyl-beta-D-thiogalactopyranoside (IPTG) to afford a finalconcentration of 0.5 mM. The incubation was continued at 37° C. for 4-6hrs or 16 hrs. Following centrifugation, the cell pellets were washed by1× PBS for three times and resuspended with homogenisation buffer [0.3 MNaCl, 0.2% IGEPAL CA-630 (Sigma, Sydney, Australia), 0.05 M sodiumphosphate buffer (pH 7), 0.25 mg/ml lysozyme], then stored frozen andlater homogenized on ice by sonication. Insoluble material was removedby centrifugation at 17,000 g for 40 min at 4° C. The fusion protein wasprecipitated from the supernatant by adding ammonium sulphate to 30%saturation. The precipitate was collected by centrifugation at 23,000 gfor 30 min at 4° C. and dissolved in 1× PBS (with 1% IGEPAL-CA 630). Thesolution was clarified by centrifugation at 23,000 g for 30 min at 4°C., and applied to a column of glutathione-Sepharose 4 FF (AmershamBiosciences, Sydney, Australia) at 4° C. with the flow-rate of 250μl/min. In order to remove the endotoxin, 50 column volumes (CV) of 1×PBS (containing 0.1% Triton X-114) was applied to remove the unboundmaterial and followed by washing with 20 CV of 1× PBS (sterile)(Reichelt P, Schwarz C and Donzeau M. Single step protocol to purifyrecombinant proteins with low endotoxin contents. Protein Expression andPurification 46: 483-488, 2006). After equilibrated with 10 CV of thefactor Xa cleavage buffer (sterile), the recombinant NC4 (rNC4) wascleaved off from the fused GST by overnight digestion with Factor Xaprotease (Amersham Biosciences or Promega) at room temperature. TherhNC4 solution migrated through the column of glutathione-Sepharose withthe elution of Benzamidine Sepharose 4 FF binding buffer (50 mMTris-HCl, 100 mM NaCl, pH 7.4), and was subjected to furtherpurification using a Benzamidine Sepharose 4 FF column (AmershamBiosciences). The flow-through rhNC4 was concentrated and desalted bywashing with dH₂O in a 5K cut-off concentrator (Agilent Technologies).Final purification was achieved by size exclusion chromatography using aSuperdex S-200 column. The size and purity of the product was determinedby SDS-PAGE analysis and western blotting. The concentration of purifiedrhNC4 was determined using the Bradford (Sigma, Sydney, Australia) orthe BCA protein assays.

Expression of Human NC4 in Yeast K. Lactis

(i) Isolation of the Collagen α1 (IX) NC4 Domain Gene

The gene of NC4 of human collagen IX (GenBank accession numberNM_(—)001851) without the 23 signal peptide was obtained byreverse-transcription PCR with RNA extracted from human chondrocytesfrom articular cartilage. Human chondrocytes were seeded in alpha-MEMmedium supplemented with 10% FCS. When the cells were confluent, theywere trypsinized and collected as a cell pellet by centrifugation. TotalRNA was extracted from the cell pellet with RNeasy® Mini Kit (QiaGen PtyLtd, Melbourne, Australia). Reverse transcription PCR was performed withSuperScript™ One-Step RT-PCR with Platinum Taq (Invitrogen, Melbourne,Australia) according to manufacturer's instructions. The upstream anddownstream primers were KL-NC451 and KL-NC431 (Table 1), respectively.cDNA synthesis was undertaken using 1 cycle of 55° C. for 30 minfollowed by 2 min, 94° C. pre-denaturation, and 40 cycles of PCRamplification (Denature: 94° C. for 30 sec; Anneal: 55° C. for 30 sec;Extend: 72° C. for 1 min). The RT-PCR products were separated by 0.8%agarose gel and purified with SNAP™ Gel Purification (Invitrogen,Melbourne, Australia).

(ii) Construction of the Vector for Expressing the Collagen α1 (IX) NC4Domain in Yeast

The K. lactis expression system (New England Biolab, USA) was applied togenerate a DNA construct for expression of the NC4 domain of the humanα1 (IX) collagen (hNC4) in yeast Kluyveromyces lactis. A group ofoligonucleotide primers (Table 1) were designed to amplify a fragment ofamino acids 24-268 of the full-length hNC4 (NCBI accession numberNP_(—)001842), which omitted the 23-amino acid signal peptide. Table 1.Primers used for construction of pKLAC1-NC4 expression vectors

TABLE 1  Primers used for construction of pKLAC1-NC4 expression vectorsName Primers KL-NC451 5′-ACTCTCGAGAAAAGAGCTGTCAAGCGTCGC-3′ KL-NC4315′-GTCAGATCTTTATCTCTCGTCGGTGGTCTG-3′ KL-NC4545′-ACTCTCGAGAAAAGAGCTGTTAAGCGTAGACCA AGATTCC-3′ KL-NC4335′-GTCAGATCTTCATTATCTCTCGTCGGTAGTCTGGCTTGGAGTAATTCTGGCTGGCAGCTCATGGCAAGT TTCTCTCCTAGGTCTCAGTGG-3′ KL-NC4385′-CTGAGATCTACCAGGTGGACCTCTTCCATCGGT AGTTTGAC-3′ GAT2-5′-CTGAGATCTGGTGCTGGTGCTATGACTAAGTTA GST53 CCTATACTAGGTTATTGG-3′ GAT2-5′-ACTGTCGACTTAGTCATTAATGATCAGATTTTG GST33 GAGGATGATCTCCACC-3′

The 5′-primer KL-NC451 contained an engineered Xho I cleavage site, andthe 3′-primer KL-NC431 contained an engineered Bgl II cleavage site.With these two primers, the hNC4 fragment without the signal peptide wasamplified by reverse transcription PCR.

The 5′-primer KL-NC454 and the 3′-primer KL-NC433 contained engineeredXho I and Bgl II cleavage sites, respectively. Different from primerKL-NC451 and primer KL-NC431, KL-NC454 and KL-NC433 provided a number ofgene mutations that changed the hNC4 gene according to yeast K. lactispreferred codons but preserved the hNC4 protein sequence unchanged.

The PCR products of hNC4 were digested by restriction enzymes andligated to the pKLAC1 expression vector (FIG. 1) at the multiple cloningsites Xho I and Bgl II. The positive inserted recombinants weresequenced to confirm that the NC4 gene insert was correct. The correctrecombinants were digested by restriction enzyme Sac II and transformedto competent K. Lactis GG799 cells. Yeast carbon base (YCB) mediumcontaining 5 mM acetamide as a source of nitrogen was used as aselective medium. Only after the Sac II fragments of the recombinantswith the target NC4 genes and amdS gene (amdS gene present in pKLAC1)have been integrated to the yeast chromosome DNA did the cells survivein this selective YCB medium.

The 5′-primer GAT2-GST53 and the 3′-primer GAT2-GST33 were designed toobtain a glutathione-S-transferase (GST) gene from the bacterial vectorpGAT-2 with some mutations for expression in yeast K. lactis. This GSTgene, which had three STOP translation codons at the 3′-terminal, wasconstructed in the vector pKLAC1 multiple cloning sites Bgl II and Sal Ito form pKLAC1-GST.

The 5′-primer KL-NC454 and the 3′-primer KL-NC438 were designed for arecombinant hNC4 that had a mutation converting the Glu²⁶⁷ to Gly. Thismutation provided a thrombin cleavage site at the C-terminal of hNC4.The recombinant hNC4 was ligated to the vector pKLAC1-GST at cloningsites Xho I and Bgl II, so that an N-terminal GST fusion protein wasobtained. After DNA sequence confirmation, the recombinant with NC4-GSTfusion protein gene was digested with Sac II and inserted to thechromosome DNA of yeast K. lactis GG799 cell, then screened by YCBselective medium for the survival colonies with integrated NC4 and amdSgenes.

(iii) Expression of the Collagen α1 (IX) NC4 Domain in Yeast K. LactisGG799 Cells

Cells from each colony that contains an integrated expression hNC4 wereharvest from an area approximately 2 mm² by scraping with a steriletoothpick or pipette tip and resuspend them in 2 ml of YPGal medium (10g Yeast Extract, 20 g Bacto™ Peptone, 2% Galactose) in a sterile culturetube. The cultures were incubated with shaking (˜250 r.p.m.) at 30° C.for a minimum of 2 days growth. Analysis of culture supernatant wasperformed each day to determine the optimum growth time to achievemaximum secretion of NC4. Larger cultures (e.g., ≧1 L) for proteinpurification were inoculated 1:100 with a starter culture grownovernight at 30° C. Samples (1 ml of each culture) were centrifuged for1 minute at 10,000 g to pellet the cells. The culture supernatant weretransferred to a fresh microcentrifuge tube and stored on ice. Thirty μlof the unconcentrated culture supernatant was applied to SDS-PAGE(NuPAGE 4-12% Tris-Bis Gel, MES buffer, Invitrogen) followed byCoomassie staining (Colloidal Blue Stain Kit, Invitrogen) and/or Westernblotting.

(iv) Partial Purification of rhNC4 Protein Expressed from Yeast K.Lactis GG799

Three-day cultures with volumes>1 L which were to used for proteinpurification were filtered with Celite-512 to remove the cells anddebris. The clear aqueous filtrates were diafiltrated and concentratedby application to a 10 KDa cut-off Tangential Flow Filtration (TFF)membrane (Millipore Ltd, Sydney, Australia). Following 2×5 volumes of 50mM sodium phosphate buffer (pH7.4) diafiltration, the culture mediumsolution was concentrated to 1-2 L and stored at 4° C. overnight orimmediately proceeded by (NH₄)₂SO₄ precipitation. Solid (NH₄)₂SO₄ wasadded to the culture medium solution to 80% saturation (0° C.). Theprecipitate was collected by centrifuge at 14,000 g, 30 min, 4° C., anddissolved in 50 mM sodium phosphate buffer (pH7.4). The proteinconcentration of purified hNC4 was determined using the Bradford or BCA(Sigma, Sydney, Australia) assays. Additional purification of thismaterial was undertaken using the same methods as described herein forthe rhNC4 prepared from E. coli.

Cryopreservation of Progenitor Cells in the Presence of VariousConcentrations of Pentosan Polysulfate (PPS)

Cells were harvested and resuspended in cold serum free culture mediumat 5.0 to 20.0×10⁶ cells/ml. An equal volume of chilled completeProfreeze®-CDM medium was added to the chilled cell suspensioncontaining 0, 10, 20, 50 and 100 micrograms/mL of PPS. The resultingfinal DMSO concentration was 7.5%, PPS concentration was 0, 5, 10, 25and 50 micrograms/mL and the final cell concentration was approximately2.5 to 10×10⁶ cells/ml. The cell mixture was aliquoted intocryopreservation ampoules (Nunc, Intermed, Denmark) and cryopreserved ina C156 Freezing Container (Thermo Scientific, Melbourne, 21 Australia)at −80° C. for a −1° C./minute controlled cooling rate. The ampouleswere then transferred to liquid nitrogen storage (−196° C.).

Thawing of Cryopreserved Samples

Cryopreserved cells were rapidly thawed for a minute in a 37° C. waterbath and transferred to a 10 mL polypropylene tube. Approximately 3 mLof appropriate media was added dropwise to the cells with constantmixing, and made up to a final volume of 10 mL. Cells were pelleted bycentrifugation at 400×g, 4° C. for 5 minutes and the supernatantaspirated. To ensure removal of residual DMSO, cells were washed inmedium and centrifuged as above. Cells were resuspended in final volumeof 10 mL and seeded in a 75 cm² flask prior to incubation in ahumidified incubator at 37° C. in the presence of 5% CO2.

Enumeration of Cells After Cryopreservation in the Presence of VariousConcentrations of Pentosan Polysulfate (PPS).

Aliquots of single-cell suspensions obtained after thawing progenitorcells from the PPS containing cryopreserved vials were diluted in anequal volume of 0.4% (w/v) trypan blue in phosphate buffered saline(PBS). The cell counts were determined using a haemocytometer (NeubauerImproved, Assistant, Germany) and a light microscope (Olympus CKX41,Japan).

The results of this experiment is shown in FIG. 1. In general, it can beseen that the addition of PPS did not have an adverse effect on thecryopreservation of the progenitor cells.

With the exception of 30 minutes, the use of 50 μg/ml PPS enhanced theviability of the progenitor cells at all time points. The use of 25μg/ml PPS either enhanced the viability or did not adversely affect theviability of the progenitor cells at all time points instead of 30minutes. The use of 10 μg/ml PPS had a beneficial effect at time point 0and then at 60 and 90 minutes. The use of 5 μg/ml PPS had a beneficialeffect on viability at 60 minutes.

Overall, it can be seen that in general, the addition of PPS did nothave an adverse effect and may enhance the cryopreservation ofprogenitor cells. The slight differences in values may be attributableto the method used to count the cells. Because the progenitor cells tendto clump, this may lead to errors which would account for the apparentlower values with 5 micrograms PPS where clumping was common.

Concentration Determined Effects of Pentosan Polysulfate (PPS) on theViability of Progenitor Cells Following Cryopreservation and ThawingUsing the MTT Assay

Murine Progenitor cells (cell lines C3H10T1/2 or ATDC5) purchased fromRIKEN Cell Bank (Tsukuba, Japan) or human immunoselected Stro-1+progenitor cells (Mesoblast Ltd, Melbourne, Australia) were seeded at avarious cell densities over the range of 1.68×10⁵-1.0×10⁶ cells into 2mL screw-capped centrifuge tubes containing in DMEM+10% FBS. In separatetubes a stock solution containing 2× the required concentrations of PPSdissolved in DMEM+20% FBS and 15% DMSO were prepared. These stocksolutions were added to the cell cultures such that the finalconcentration of PPS was half that of the stock concentration and of FBSwas 10% and DMSO was 7.7%. Over several independent experiments thefinal concentrations of PPS in the final solutions ranged from 0.0-100ug/ml (see figures for details). All concentrations of PPS used wereexamined in triplicated. Cells and the cryopreservation solutionscontaining the PPS were mixed gently for 5 mins and stored in liquidnitrogen. After 3 days, the tubes were removed from liquid nitrogen andthawed in 37° C. water bath and allowed to stand at ambient temperaturefor about 15 min. The cells were centrifuged down at 200 g for 5 min andthe supernatant discarded. The cells were washed with 900 ul of DMEMwithout phenol red, and then centrifuged down at 200 g for 5 min. Thecells were resuspended in 500 ul of 1 mg/ml MTT solution in DMEM andincubated in 37° C. for 3 hours then centrifuged at 6000 g for 5 min.300 ul of DMSO was added to each tube to dissolved the dye crystals. 90ul×3 from each tube were transferred to a 96-well plate and theabsorbance at 540 nm determined.

FIG. 2 shows a bar graph showing the viability of different numbers ofmurine ATDC5 progenitor cells suspended in cryogenic media containing7.5% DMSO and various concentrations of Pentosan Polysulfate (PPS) afterbeing subjected to freeze-thawing cycle. Cell viability was determinedusing the MTT assay.

FIG. 2 shows that the viability of the progenitor cells were enhanced bythe presence of PPS at all concentrations. At 100 μg/ml PPS theviability was enhanced at cell counts of 0.25 million, 0.5 million andat 1.0 million cells. The same is seen with 250 μg/ml PPS. For 500 μg/mlPPS, enhancement is seen for 0.25 million and 0.5 million cells. For 1million cells, a concentration of 500 μg/ml PPS did not appear toenhance viability but was not detrimental to viability.

By extrapolation of these data it is may be assumed that a dosage of 100million of chondroprogenitor cells together with 25-50 mg PPS whensubjected to a freeze-thawing cycle in an appropriate cryoprotectivemedium would maintain the viability of the cells to a level foracceptable administration to a patient in need of such therapy.

FIG. 3 shows the effects of different concentrations of Sodium PentosanPolysulfate (PPS) on human progenitor cell viability followingcryopreservation at −180° C. and thawing as determined using the MTTassay. Data shown=Means±SD*=p<0.05 relative to control values.

When cryopreserving 168,000 cells, the presence of PPS improved theviability of the cells, particularly at 1 and 2.5 μg/ml PPS. With350,000 cells, it can be seen that in general, viability of theprogenitor cells is not adversely affected by the presence of PPS. Insome cases, viability of the cells is enhanced after thawing.

By extrapolation of these data it is may be assumed that a dosage of 100million human progenitor cells together with 30 mg PPS when subjected toa freeze-thawing cycle in an appropriate cryoprotective medium wouldmaintain the viability of the cells to a level for acceptableadministration to a patient in need of such therapy.

Effects of Pentosan Polysulfate (PPS) on Human Progenitor CellsApoptosis Induced by the Addition of a Combination of IL-4 IFN-Gamma asDetermined by Flow Cytometry.

Human progenitor cells were plated in serum-free media supplemented withPPS at concentrations of 0, 1, 2, 5 and 10 micrograms/mL. Progenitorcell apoptosis was induced by the addition of a combination of 30 ng/mlIL-4 30,000 U/ml IFN gamma. Following 5 days culture, cells wereharvested by trypsinisation and viabilities assessed by Annexin Vstaining as previously described (Kortesidis A, A Zannettino, SIsenmann, S Shi, T Lapidot and S Gronthos. (2005). Stromal-derivedfactor-1 promotes the growth, survival, and development of human bonemarrow stromal stem cells. Blood 105:3793-3801).

The results for this experiment is shown in FIG. 6. It can be seen thatcell apoptosis is reduced for all concentrations of PPS with the bestresults seen for 10 μg/ml. These results indicate that the addition ofPPS into the cryopreservation medium will reduce apoptosis on thawing.

The experiment was undertaken in a 96 well plate containing 50,000progenitor cells/well. Without wishing to be bound by theory it isbelieved that apoptosis and activation of the stress protein cascade isa recognised sequence of cell freeze-thawing and therefore the abilityof PPS to significantly reduce this process must be of benefit to theuse of these cells following cryopreservation and thawing as required inmost cell based medical procedures.

Effects of Sodium Pentosan Polysulfate (PPS) Alone or in Combinationwith rhNC4 on the Biosynthesis of Proteoglycans (Measured as ³⁵S-GAGs)by Progenitor Cells Grown in Monolayer Cultures

Murine Progenitor cells (cell lines C3H10T1/2 or ATDC5) purchased fromRIKEN Cell Bank (Tsukuba, Japan) or human immunoselected Stro-1+Progenitor cells (Mesoblast Ltd, Melbourne, Australia) were seeded into50 mL plastic culture flasks containing 1:1 high glucose DMEM/Ham's F-12medium (Invitrogen) supplemented with 10% FBS (Sigma) and incubated at37° C., in 5% CO₂. After confluence was reached, progenitor cells werereleased by trypsinization and harvested by centrifugation. Cells wereinoculated to 96-well or 24-well plates at a density of 3×10⁴ cells or2×10⁵ cells per well. After 48-hour incubation at 37° C., 5% CO₂confluent monolayers were normally established. The medium was thenchanged to defined medium, which contained different concentrations ofPPS with and without rhNC4 and 5 μCi/ml of ³⁵S-H₂SO₄ (PerkinElmer, USA).The experiments were generally terminated after 48 hours incubation whenproteoglycan biosynthesis was determined by measuring the incorporationof ³⁵SO₄ into the glycosaminoglycans (³⁵S-GAGs) as described below.

(A) 96-well plates: Cultures were subjected to proteolytic digestionwith papain to release glycosaminoglycans. The papain stock solutionused contained 2.5 mg/ml papain (Sigma), 7.9 mg/ml cysteine-HCl (Sigma)in papain digest buffer (0.1M NaAc, 5 mM EDTA, pH6.0). 50 μl of papainstock solution was added to each well. The plate was sealed usingplastic sheeting and incubated at 65° C. for 2 hours. On termination,aliquots of 50 μl digested solution-per well were collected for DNAfluorometric assay using Hoechst 33258 Dye and the method described byKim et al (Kim Y J, Sah R L Y, Doong J-Y H, Grodzinsky A J, Fluorometricassay of DNA in cartilage explants using Hoechst 3358. Anal Chem. 1988;174: 168-176).

The ³⁵SO₄-GAGs in the remained solution (200 ul) were precipitated withCetyl Pyridinium Chloride (CPC) (Sigma). Briefly, to each well was added20 μl of 5% CPC, followed by 10 μl of 1 mg/ml Chondroitin sulfate A(CSA, Sigma) as a co-precipitant. The ³⁵S-GAG-CPC complexes werecollected by vacuum filtration using a cell harvester (Skatron 7021).The filters were then air-dried and discs punched into scintillationvials. Following addition of 3 ml scintillation liquid and vortexing theradioactivity of the ³⁵S-GAG-CPC complexes were in the samples weremeasured by scintillation counting (PerkinElmer, USA) and recorded asDPM/sample. The data then was then normalised for DNA and expressed as³⁵-S-GAGs/μg DNA.

(B) 24-well plates: After 48 hours incubation in the defined media, themedia per well (containing soluble ³⁵S-labelled proteoglycans) wasseparated from the cells and transferred to a 2.0 ml microfuge tube. Themonolayer cells remaining in the wells were detached by trypsinization,and then separated from the supernatant (containing matrixproteoglycans) by centrifugation at 350 g for 5 min. The supernatant wascollected and combined with the medium. 200 μl of the medium andsupernatant mixture was transferred to a 96-well plate and subjected topapain digest to release glycosaminoglycans. After papain digestion, 20μl of 5% CPC was added to each well to precipitate the 35S-GAG followedby 10 μl of 1 mg/ml CSA as a carrier. The 35S-GAG-CPC complexescollected through the fibre filter and the radioactivity of 35S-GAG wasmeasured using the liquid scintillation analyser as describe above. Thecells were extracted with the RNA reagent and aliquots used to determinegene expression and/or the DNA content was measured by the fluorometricassay and the results expressed as ³⁵-S-GAGs/μg DNA.

FIG. 4 shows the Effects of Pentosan Polysulfate on Human Progenitorcell Proliferation.

Primary human progenitor cells were cultured in 24 well plates in growthmedia supplemented with PPS at the indicated concentrations. At varioustime intervals (day 1,3,6), the growth media was removed and replacedwith phenol red free media containing the tetrazolium salt WST-1 for 2hours at 37° C./5% CO₂. WST-1 is cleaved by mitochondrial dehydrogenasein viable cells to produce a formazan dye that can be detected using anELISA plate reader at a wavelength of 450 nm. Absorbance at 450 nm foreach time point is shown for all concentrations of PPS. A statisticallysignificant increase in proliferation was observed on day 6 atconcentrations of PPS in excess of 1 μg/ml (*p<0.01, ANOVA). FIG. 4shows that progenitor cell viability was enhanced relative to progenitorcells frozen in cryo-preservation media not containing the polysulfatedpolysaccharide.

FIG. 9 shows the concentration dependent effects of Sodium PentosanPolysulfate (PPS) on Murine Progenitor cell (C3H10T1/2) biosynthesis ofProteoglycans (PGs) and DNA content when grown in monolayer cultures.The data shown is Means±SD.

It can be seen that at all concentrations of PPS the biosynthesis ofproteoglycans is increased. This shows that the use of polysulfatedpolysaccharides can induce differentiation, especially chondrogenesis atall concentration ranges. The best result is seen for 5-10 μl/ml, with10 μl/ml being best with regards to PG synthesis and 5 μl/ml and 10μl/ml being best with regard to DNA content.

FIG. 10 shows a bar graph of the concentration dependent effects ofPentosan Polysulfate (PPS) on DNA synthesis by murine Progenitor cells(C3H10T1/2 cells) grown in monolayer cultures for 2 days as determinedby the incorporation of ³H-Thymidine into macromolecular DNA.

FIG. 11 shows a bar graph of the concentration dependent effects ofPentosan Polysulfate (PPS), on the biosynthesis of Proteoglycans PGs) asdetermined by the incorporation of radioactively labelled sulfate intothe sulfated glycosaminoglycans (³⁵S-GAG) of PGs after 2 day monolayercultures of human progenitor cells. The data was expressed as ³⁵S-GAGradioactivity as decays per minute (DPM) normalised to DNA content.*=p<0.05; **=p<0.005; ***=p<0.0005.

FIG. 21 shows a bar graph of the concentration dependent effects ofrhNC4 (batch PBA-1202P) expressed by K. lactis, in the absence(maintenance media, MM) and presence of insulin (10 micrograms/mL)(differentiation media, DM), on the biosynthesis of Proteoglycans (PGs)as determined by the incorporation of radioactively labelled sulfateinto the sulfated glycosaminoglycans (³⁵S-GAG) of PGs after 3 dayculture with Murine ATDC5 progenitor cells. The data was expressed as %change relative to control cultures that contained no rhNC4. P<0.05 wasstatistically significant relative to control cultures.

FIG. 24 shows the results for the combination of PPS and NC4. It can beseen that the effect of PPS increases with concentration. It can also beseen that this effect is enhanced in the presence of increasingconcentrations of NC4.

The combination of 0.5 μl/ml of NC4 and 5 μl PPS showed a statisticallysignificant increase in proteoglycan synthesis. In addition, thecombination of 1 μl/ml NC4 and 2 μl/ml and 5 μl/ml PPS showed astatistically significant increase in proteoglycan synthesis.Furthermore, the combination of 2 μl/ml NC4 together with 1 μl/ml, 2μl/ml or 5 μl/ml PPS or the combination of 5 μl/ml NC4 together with 1μl/ml, 2 μl/ml or 5 μl/ml PPS all showed a statistically significantincrease in proteoglycan synthesis.

The use of 5 μl/ml NC4 or 5 μl/ml PPS showed the best results with thecombination of 5 μl/ml NC4 and 5 μl/ml PPS being best.

Effects of Sodium Pentosan Polysulfate (PPS) Alone or in Combinationwith rhNC4 on the Biosynthesis of Proteoglycans (Measured as ³⁵S-GAGs)by Progenitor Cells Grown in Pellet Culture

Murine Progenitor cells (cell lines C3H10T1/2 or ATDC5) purchased fromRIKEN Cell Bank (Tsukuba, Japan) or human immunoselected Stro-1+Progenitor cells (Mesoblast Ltd, Melbourne, Australia) were seeded in 2ml screw-capped sterile centrifuge tubes and the total volume made-up to1 ml with DMEM-High glucose medium containing 10% FBS. Progenitor cellswere then centrifuged at 500 g for 10 min in a swing-out rotor at roomtemperature. The screw caps were loosened and the tubes placed in anincubator at 37° C., in a 5% CO₂/95% air moist atmosphere. The pelletsgenerally formed within 24 hours. Medium was changed daily in the firsttwo days, then once every 2-3 days thereafter. On day 5 the medium wasremoved and to each tube was added 1 ml with DMEM-High glucose mediumcontaining 10% FBS, 5.0 μCi/ml ³⁵S-H₂SO4 and various concentrations ofPPS (0, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 20.0, μg/ml) (FIG. 12-15) orrhNC4 (0, 0.1, 0.5, 1.0, 2,0 μg/ml) (FIG. 22). Triplicate cultures wereused for all concentration of drugs. The screw caps were loosened andthe tubes placed in an incubator at 37° C., in a 5% CO₂/95% moist airatmosphere for 3 days. On day 6, 200 μl of stock papain solution [2.5mg/ml papain, 7.9 mg/ml L-Cystein in papain buffer (0.1 M NaAC, 5 mMEDTA, pH6.0)] was added to each tube. The tubes were tightly capped andincubated at 65° C. for 2 hours. After papain digestion, 4 replicates of200 μl of papain digested solution were separately transferred to a96-well micro-titre plate (200 μl/well). The remaining solution was usedfor the DNA fluorometric assays which were undertaken in triplicate asdescribed above. Biosynthesis of ³⁵S-GAGs was determined as describedfor the monolayer cultures and the results expressed as ³⁵-S-GAGs/μgDNA.

FIG. 22 shows a bar graph of the concentration dependent effects ofrhNC4 (batch PBA-1202P) expressed by K. lactis, in the absence(maintenance media, MM) and presence of insulin (10 micrograms/mL)(differentiation media, DM), on the biosynthesis of Proteoglycans PGs)in pellet cultures of ATDC5 cells. Data is shown as the % of controlstaken to be 100%.

Similar experiments were undertaken using Heparin (Sigma, Sydney,Australia) in place of PPS. These results are seen in FIG. 14. Theheparin regulates differentiation by suppressing chondrogenesis of theprogenitor cells.

Effects of Sodium Pentosan Polysulfate (PPS) Alone or in Combinationwith rhNC4 on the Biosynthesis DNA (Measured as ³H-ThymidineIncorporation) by Progenitor Cells Grown in 6 Day Pellet Cultures

Murine Progenitor cells (cell lines C3H10T1/2 or ATDC5) purchased fromRIKEN Cell Bank (Tsukuba, Japan) or human immunoselected Stro-1+Progenitor cells (Mesoblast Ltd, Melbourne, Australia) were seeded at adensity of 3×10⁵ cells 2 ml screw-capped centrifuge tube and topped upto 1 ml with DMEM-High (+10% FBS). Cells were centrifuged down at 500 gfor 10 min in a swing-out centrifuge in room temperature then wereincubated at 37° C. in a moist atmosphere of 5% CO2/95% air for 24hours. The pellets were observed to be established over this time.Medium was changed every day in the first two days, then once every 2-3days thereafter. On day 3, the medium was removed, to each tube wasadded in 640 microL of DMEM (+10% FBS), 80 microL of 50 mCi/ml3H-Thymidine and 80 microL of rhNC4 (0, 1, 2.5, 5, 10, 25microgramsg/ml). (FIG. 23)

All assays were undertaken in triplicated. After 3 days (66 hrs), thesupernatant medium was removed and saved for the HA ELISA. 100 microL of1 mg/ml collagenase (dissolved in DMEM medium) was added to each pellettube. Tubes were incubated at 37° C. in a shaker at 180-200 rpm for 3.5hrs. The collagenase digest was transferred to a 96-well plate such thatthe contents of each tube was divided into 4 wells. To each well wasadded in 200 microL dH2O and the plate stored in −20° C. for lateranalysis. The collagenase digests were thawed and DNA was collected witha cell harvestor and the filter discs placed in a scintillation tube.Scintillation cocktail liquid (3 mL) was added and vortexed about 1 min.The radioactivity of ³H-DNA in these samples was determined using aβ-scintillation counter.

Effects of Sodium Pentosan Polysulfate (PPS) Alone or in Combinationwith rhNC4 on the Biosynthesis of Proteoglycans (Measured as ³⁵S-GAGs)by Progenitor Cells Grown in Micromass Cultures

The technique used was based on that described by (Denker A E, Haas A R,Nicoll S B and Tuan R S. Chondrogenic differentiation of murineC3H10T1/2 multipotential progenitor cells: I. Stimulation by bonemorphogenetic protein-2 in high density micromass cultures.Differentiation (1999); 64: 67-76). Briefly murine Progenitor cells(cell lines C3H10T1/2 or ATDC5) purchased from RIKEN Cell Bank (Tsukuba,Japan) or human immunoselected Stro-1+ Progenitor cells (Mesoblast Ltd,Melbourne, Australia) Ten microlitres (10 μl) of a suspension of theprogenitor cells (1×10⁷ cells/ml in Ham's F12 medium+10% FBS) wereapplied to individual wells of a 24-well plates. After incubation at 37°C. in a humidified atmosphere of 5% CO2/95% air for 2-3 hours, 900 μl ofHam's F12 medium (10% FBS) and 100 μl of PPS solution in the same mediumwere added slowly into the wells to afford final concentrations of PPSin each of 0, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 20.0 μg/ml alone or incombination with rhNC4 (0, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 25.0 μg/ml).Each drug concentration was repeated in triplicate. The cells weremaintained in culture for up to 10 days. The medium with/without drugswas changed every 3 days but 24 hours before termination of theexperiment 80 μl of 50 μCi/ml ³⁵S-H₂SO₄ was added to achieve a final³⁵SO₄ concentration of 5 μCi/ml. The following day, the cells in one ofthe plates were trypsinized with 150 μl/well of 2.5% trypsin andseparated from the medium by centrifugation at 800 g for 10 minutes,washed with 500 μl of 1× PBS and stored in liquid nitrogen for RNAand/or DNA extraction and analysis. Media and supernatants were combinedin one tube for each concentration of PPS used. To the tubes was added,200 μl of 5× Papain solution [2.5 mg/ml Papain, 7.9 mg/ml L-Cysteine inPapain buffer (0.1 M NaAC, 5 mM EDTA, pH6.0)] and the solution incubatedat 65° C. for 2 hours. After papain digestion, Four aliquots of 200 μlof digested solution were transferred to a 96-well plate (200 μl/well).The ³⁵S-GAG released by the digestion step were separated from the free³⁵SO4 by adding 20 μl 5% CPC and 10 μl 1 mg/ml CSA with gentle shakingat 300 rpm RT for 20-30 min to precipitate the ³⁵S-GAG-CPC complexes.The precipitates were collected by filtered through glass fibre filtersusing a cell harvester. The filters were then air-dried and discspunched to scintillation vials. 3 ml/vial scintillation cocktail liquidwas added and vortexed for 30-45 sec. The radioactivity of ³⁵S-SO₄incorporated the ³⁵S-GAG-CPC complexes was measured by scintillationcounting.

Immunochemical Stain of Micromass Cultures of Progenitor Cells for TypeII Collagen

Micromass cultures of progenitor cells were established in 24-wellculture plates at a starting density of 8×10⁴ cells/micromass in 1 ml ofDMEM (+10% FBS) with/without various concentrations of PentosanPolysulfate as described above. On Day-5 and Day-10 of culture, mediawas removed and cultures were fixed with Histochoice MB (Amresco, Solon,Ohio, USA) for 20-30 min at RT. The fixed cultures were washed twice inPBS (5 min each time); p washed in PBS twice (5 min each time); bl for20 min at RT then washed in PBS for 5 min. The plates were thenincubator. After rinsing, first with a gentle stream of PBS, followed bywashing 3 times (5 min each time) with PBS, the wells were blocked withP5 minutes. To deIgG rinsing with a gentle stream of PBS then 3× washing(5 min each time) with PBS, to each micromass culture was added 200 μlof 1× (BCIP/Buffer+NBT) and the plates incubated in the reaction wasstopped when the purple color first became established in the section bywashing in running water. Plates and wells were then photographed with adigital camera and the images analysed by using Image J® software(http://rsb.info.nih.gov/ij/) on a personal computer.

FIG. 16 shows a bar graph showing the concentration dependent effects ofPPS on proteoglycan synthesis by murine progenitor cells (C3H10T1-2) inmicromass cultures for 6 days and 9 days. PPS was included in the media(Ham's F12+10% FCS) and was changed every 48 hours. ³⁵S-SO₄ was added 24hours before culture termination. Synthesis normalized to DNA content.*P<0.05, **P<0.005, ***P<0.0005.

Highly significant stimulation of uptake of 35S into newly synthesizedPG was observed over the concentration range of 1-20 micrograms/mL inthe this cell line. (FIG. 16). Moreover, after nine days in micromasscultures a 100% stimulation was obtained at 1 microgram/mL of PPS (FIG.16).

FIG. 17 shows bar graphs showing the concentration dependent effects ofPPS on proteoglycan synthesis by human Progenitor cells in micromasscultures for 5 days. Data is presented as 35S-GAG radioactivity and as apercentage of control taken as 100%. *P<0.05 relative to control.Increased proteoglycan synthesis was seen at concentration rangesbetween 0.5-10 μg/ml.

Human Progenitor cells also differentiated to chondrocytes in micromasscultures when incubated in the presence of PPS. However in the 5 daycultures a maximum stimulation of PG synthesis of 30% was obtained withPPS concentration of 2.5 micrograms/mL (FIG. 17).

FIG. 18 shows a bar graph showing the Pentosan Polysulfate (PPS)concentration dependent stimulation of type II collagen production byhuman Progenitor cells in micromass cultures for 10 days as determinedby scanning and analysis of the immuno stained micromass cultures shownin B. Increased Type II Collagen production was seen at concentrationranges between 0.5-10 μg/ml with the best results seen for 5 μg/ml.

These results indicate that PPS induces the progenitor cells todifferentiate into chondrocytes, as evidenced by increased synthesis ofboth proteoglycans and Type II Collagen.

Similar experiments were undertaken using Hyaluronic acid (SuperArtz(SKK, Tokyo, Japan)) and Dextran polysulfate (MW=5000) (Sigma, Sydney,Australia) in place of PPS. These results are shown in FIG. 19 whichshows bar graphs showing the concentration dependent effects of (A)Hyaluronan (HA) (Supartz™) and (B) Dextran Polysulfate on proteoglycansynthesis by human Progenitor cells in micromass cultures for 5 days.Data is presented as 35S-GAG radioactivity and as a percentage ofcontrol taken as 100% or as DPM/ug DNA. *P<0.05 relative to control. Itcan be seen that HA appeared to increase synthesis of proteoglycans butnot strongly. In contrast, dextran sulfate downregulated thedifferentiation of progenitor cells as is evidenced by a concentrationdependent reduction in the production of proteoglycans.

Concentration Effects of Sodium Pentosan Polysulfate (PPS) on theBiosynthesis DNA (Measured as ³H-Thymidine Incorporation) by ProgenitorCells Grown in 8 Day Micromass Cultures

Ten μl of progenitor cells (7×10⁶ cells/ml) were seeded into each wellof a 24-well culture plate. After incubation at 37° C. in moist 5%CO2/95% air for 2-3 hours, 900 μl of DMEM-High medium (+10% FBS) andindicated concentrations PPS dissolved in DMEM-High medium were addedslowly to the wells. The final PPS concentration were 0, 0.1, 0.5, 1.0,2.5, 5.0, 10.0, 20.0 μg/ml, respectively. Every PPS concentration wasestablished in triplicate. The cultures were allowed to proceed for 3days. On day 4, to each well of the 24-well plate was added 640 μl ofDMEM-High (+10% FBS), 80 μl of 10× PPS solution and 80 μl of 10 μCi/ml³H-Thymidine solution to produce a final ³H-Thymidine concentration of 1μCi/ml and final PPS concentrations as indicated. The cultures were thenincubated in 37° C., 5% CO2 for a further 22 hours, the medium wasremoved and 200 μl of 1 mg/ml Collagenase solution [Collagenase buffer:66.7 mM NaCl, 6.7 mM KCl, 4.8 mM CaCl2.2H2O, 10 mM HEPES (pH7.4)] wasadded to each well. The plate was incubated at 37° C. for 2.5 hours torelease the cells. Every half an hour, the plate was shaken gently byhand. After collagenase digestion, the cells and digestion solution werecentrifuged at 500 g for 5 min. The supernatant was removed. The cellswere gently mixed with 200 μl of TE buffer and lysed by freezing-thawingtwice. After lysis, 200 μl more TE buffer was added to the cells withmixing. Aliquots of the cell lysate was applied to a 96-well plate asfour repeats (100 μl each well). The ³H-DNA in the cell lysate wascollected using glass fiber filters and a cell harvester. The filterswere then air-dried and punched to scintillation vials. 3 ml/vialscintillation cocktail liquid was added in and vortexed for 30-40 sec.The radioactivity of ³H incorporated into the DNA of proliferating cellswas measured by scintillation counting.

The results are shown in FIG. 5 which shows increased proliferation ofprogenitor cells in combination of PPS. It can be seen that allconcentrations of PPS increased proliferation with the best resultsbeing seen at concentrations of 1 and 2.5 μg/ml.

Concentration Effects of Sodium Pentosan Polysulfate (PPS on theBiosynthesis of Hyaluronan (HA) by Progenitor Cells by Measuring theIncorporation of ³H-Glucosamine into HA

Human immunoselected Stro-1+ Progenitor cells (Mesoblast Ltd, Melbourne,Australia) were established in micromass cultures in 24 well platesusing the method described above but seeding the progenitor cells at adensity of 7×10⁵ cells/well. After 24 hours in culture the media waschanged and replaced with DMEM culture medium containing 10% FCS and 25μg/ml gentamicin containing PPS (Bene-Arzneimittel, Munich, Germany) atconcentrations of (0.0, 0.5, 1.0, 2.5 micrograms/mL) which had beensterilised through a 0.22 μm filter. The cultures were incubated in 5%CO₂/95% moist air at 37° C. for 8 days with media changes containing theindicated concentrations of PPS every 3 days. On the 8^(th) day³H-glucosamine was added to culture medium containing the indicatedconcentrations of PPS to provide a solution containing 1.0 μCi/ml whichwas added to each well. The plates were incubated for a further 24 h. Attermination of the cultures on day 9, media was collected into 5 mlcapped tubes and stored at 4° C. prior to size exclusion chromatographyas described below.

Isolation and Quantitation of ³H-Hyaluronan (³H-HA) in Culture MediaUsing Gel Filtration Chromatography

Two aliquots of 0.5 ml from each media sample were labelled A and B. 20μl of 1 M acetic acid, pH 6.0 was added to all aliquots. 50 μl ofreaction buffer (20 mM Na-acetate and 0.15 M NaCl, pH 6.0) was added toaliquot A and 50 μl of 5 TRU Streptomyces hyaluronidase (HYALASE) inreaction buffer was added to aliquot B. All samples were incubated at60° C. for 3 h followed by boiling for 5 min to inactivate the addedhyaluronidase. The samples were stored at −20° C. prior to gelfiltration.

A Gel filtration column prepacked with Superdex-S200 was used to isolateand identify ³H-HA and ³H-PGs in culture media. Media samples wereroutinely centrifuged at high speed on a bench Microfuge for 10 minimmediately before loading to the column. Samples (200 μl of each) wereinjected into the column through sample loop and the column was elutedwith PBS buffer (0.15 M NaCl, 0.05 M Na₂PO₄, pH 7.2) at the flow rate of0.2 ml/l min. The column eluent was collected at 1.0 ml/fraction fortotal of 186 fractions and radioactivity was determined using aβ-scintillation counter.

These results are shown in FIG. 27. Analysis of the area under thechromatographic profiles before and after digestion with theStreptomyces hyaluronidase (HYALASE) for control cultures containing noPPS showed that 14.3% of the ³H-glucosamine was incorporated into HA and85.7% into the PG subunits. As is evident from the profiles shown in 27Athe molecular size of the PG-HA aggrecan complex was larger than the PGmonomers which become liberated when the HA is digested away. Of theconcentrations of PPS examined only 0.1 micrograms/mL and 1.0micrograms/mL showed a substantial increases in levels of newlysynthetised ³H-HA. in the culture media. The proportion of radioactivitypresent in the post digestion void volume fractions of PG monomers being50.4% showing that 49.6% was incorporated into HA for 1 microgram/mLFIG. 27C) and 35.5% for 0.1 micrograms/mL (profile not shown). With thelower concentration of PPS (FIG. 27B) 15.1% of radioactivity was foundin the HA fractions, while the higher concentration of 2.5 micrograms/mLonly incorporated about 11% of ³H-glucosamine into HA. Although thesedata suggest that maximum synthesis of HA by progenitor cells inmicromass cultures occurred at the PPS concentration of 1.0micrograms/mL, the ³H-HA levels remaining in the micromass extracellularmatrix have yet to be determined. Since parallel studies describedherein have shown that PPS stimulates chondrogenic differentiation ofprogenitor cells and the formation of cartilage proteoglycans, theextracellular matrix surrounding the cells may represent a richer sourceof the newly synthetised HA in the form of a component of the PGaggrecan complex. Nevertheless, this is the first report whichdemonstrates that PPS stimulates the biosynthesis of HA by culturedprogenitor cells.

Concentration Effects of rhNC4 (Batch PBA-1202p) Alone and inCombination with Sodium Pentosan Polysulfate (PPS on the Biosynthesis ofHyaluronan (HA) by Progenitor Cells by Measuring the Incorporation of³H-Glucosamine into HA

Murine Progenitor cells (cell lines C3H10T1/2 or ATDC5) purchased fromRIKEN Cell Bank (Tsukuba, Japan) or human immunoselected Stro-1+Progenitor cells (Mesoblast Ltd, Melbourne, Australia) will beestablished in micromass cultures as described above or seeded at1.5×10⁵ cells/well in 6-well culture plates and allowed to attach for 24h before addition of compounds. Various concentrations of rhNC4preparations alone and in combination with PPS (Bene-Arzneimittel,Munich, Germany) will be prepared in DMEM culture medium containing 10%FCS and 50 μg/ml gentamicin at double the concentration required in thecultures, sterilised through a 0.22 μm filter and then serially dilutedto give final concentrations of the drugs required for each experiment.Aliquots of each of the test solutions will be added to each well of24-well culture plates. Stock ³H-glucosamine will be diluted in culturemedium to give a 1.0 μCi/ml solution which will be immediately added toeach well. The plates will be incubated for a further 24 h. At culturetermination, media will be collected into 5 ml capped tubes and storedat −20° C. for ³H-HA analysis. Cells will be released by trypsinizationand centrifuged with washing. Trypsin mobilized radioactivelylabelled-HA and washing will be analysed as for the media and the cellswere then used for extraction of RNA and evaluation of gene expressionas described below.

Isolation and Quantitation of ³H-Hyaluronan (³H-HA) in Cultures UsingGel Filtration Chromatography

Two aliquots of 0.5 ml from each media sample will be labelled A and B.20 μl of 1 M acetic acid, pH 6.0 will be added to all aliquots. 50 μl ofreaction buffer (20 mM Na-acetate and 0.15 M NaCl, pH 6.0) will be addedto aliquot A and 50 μl of 5 TRU Streptomyces hyaluronidase in reactionbuffer will be added to aliquote B. All samples will be incubated at 60°C. for 3 h followed by boiling for 5 min to inactivate the addedhyaluronidase. The samples will be store at −20° C. prior to gelfiltration.

Gel filtration columns prepacked with either Superose 6 or Superdex-S200will be used to isolate and identify ³H-HA in culture media. Mediasamples will be routinely centrifuged at high speed on a bench Microfugefor 10 min immediately before loading to the column. Samples (200 μl ofeach) will be injected into the column through sample loop and thecolumn will be eluted with PBS buffer (0.15 M NaCl, 0.05 M Na₂PO₄, pH7.2) at the flow rate of 0.2 ml/l min. The column eluent will becollected at 0.5 ml/fraction for total of 46 fractions and radioactivitywill be determined using a β-scintillation counter.

This experiment will show a concentration dependent stimulation of HAsynthesis with optimum effects over the range of 1-5 micrograms/mL ofPPS alone and rhNC4 of 5-25 micrograms/mL. In combination 2micrograms/mL with 5-25 micrograms/mL of rhNC4 will show synergy.

Concentration Effects of rhNC4 (Batch PBA-1202p) Alone and inCombination with Sodium Pentosan Polysulfate (PPS on the Biosynthesis ofHyaluronan (HA) by Progenitor Cells Using a ELISA

Murine Progenitor cells (cell lines C3H10T1/2 or ATDC5) purchased fromRIKEN Cell Bank (Tsukuba, Japan) or human immunoselected Stro-1+Progenitor cells (Mesoblast Ltd, Melbourne, Australia) will be seeded at2×10⁵ cells/well in 24 well culture plates and maintained in 1 mlDMEM/Ham's F-12 medium (Invitrogen) supplemented with 10% FBS (Sigma)and incubated at 37° C. in a 5% CO2/95% moist air atmosphere until thecells reached 80% confluence. The media will then be replaced withDMEM/Ham's F-12 containing various concentrations of the rhNC4preparations alone and in combination with pentosan polysulfate(Bene-Arzneimittel, Munich, Germany) and the cultures will be maintainedat 37° C., in 5% CO₂ for a further 24 hours. The media in each well willbe separated from the cells and transferred to a 2.0 ml microfuge tube.The monolayer cells remaining in the wells will be detached bytrypsinization and then separated from the supernatant by centrifugationat 350 g for 5 min. The supernatant will be collected and combined withthe medium. The combined medium and supernatant mixture (200 μl) will beassayed for hyaluronan content using the HA-ELISA as described above.The supernatant from the cell trypsinization will be boiled to denatureand inactivate the enzyme and also assayed for HA content using theELISA. The HA of these fractions will be considered to represent the HAcontent of the extracellular matrix (ECM).

The results of this experiment will confirm the results found in FIG.27. The ELISA will demonstrate HA production by progenitor cells in thepresence of low doses (including 1-5 microgram/ml) of polysulfatedpolysaccharides.

Effects of Sodium Pentosan Polysulfate (PPS) on the Differentiation ofHuman Progenitor Cells in an Osteogenic Media Using in VitroMineralisation Assays.

The conditions necessary for the induction of human progenitor cells todevelop mineralised bone matrix in vitro have been previously described(Gronthos S, A C Zannettino, S J Hay, S Shi, S E Graves, A Kortesidisand P J Simmons. (2003). Molecular and cellular characterisation ofhighly purified stromal stem cells derived from human bone marrow. JCell Sci 116:1827-1835). The osteoinductive media consists of alpha-MEMmedia supplemented with 2% (v/v) FCS, 1.8 mM KH2PO4, 10-7 dexamethasonesodium phosphate, 50 IU/ml penicillin, 50 μg/mL streptomycin, 1 mMSodium Pyruvate, 100 μM L-Ascorbic acid 2-phosphate, 2 mM L-glutamineand 10 mM HEPES buffer.

Human progenitor cells were seeded into 96-well plates at 8×10³ cellsper well and allowed to reach ≧90% confluence prior to the addition ofosteoinductive media containing nominated concentrations of PPS (0.0,1.0, 5.0, 10 micrograms/mL). Cells were cultured at 37° C. in thepresence of 5% CO2 for the indicated period. The osteoinductive culturemedia containing fresh compound was changed twice weekly for a period of4 weeks.

The results from this experiments can be seen in FIG. 7A. It shows thatthe presence of PPS suppressed differentiation into osteocytes,particularly at 1 and 10 micrograms PPS/mL

Effects of Sodium Pentosan Polysulfate (PPS) on the Differentiation ofHuman Progenitor Cells in an Osteogenic Media—Analysis of in VitroMineral Content

Total mineral content per well in the above cultures was assessed bymeasuring the calcium levels per total DNA in each well. Cell cultureswere washed three times with Ca+ and Mg+ free PBS and left to solubilizeovernight in 0.6M hydrochloric acid (100 μL per well). Theacid-solubilized mineral 23 was transferred to a new 96-well plate andreacted with o-cresol-phthalein-complexone (Thermal ElectronCorporation, USA) to form a purple dye that was measured at 570 nm usingan EL 808 Ultra Microplate Reader. The intensity of the purple dye isdirectly proportional to the calcium concentration in each well.Absolute calcium concentration was extrapolated from a calcium standardcurve according to the manufacturer's recommendations. Following this,the acid-solubilized cultures were rinsed three times with Ca2+ and Mg2+free PBS and incubated in a 100 μLs of solution of 100 μg/mL ProteinaseK at 37° C. for 2 hours. Digested samples were vigorously pipetted and50 μL of each well was transferred to a well of a nonfluorescent assayplate, containing 150 μL diluted Hoechst 33258 (2 μg/mL) in DNA assaybuffer (2M NaCl and 50 mM Sodium Phosphate). Absolute absorbance wasdetermined by measuring against a series of DNA standards at 350 nm byLS55 Luminescence Spectrometer (Perkin Elmer).

The results from this experiments can be seen in FIG. 7B. The fact thatPPS cultures all appear the same as the media indicated that nocalcified deposits are present. Normally mineralized deposits stainedpositively with the Alizarin Red reagent and are formed within 4 weeksof culture of progenitor cells under osteoinductive conditions.

Effects of Sodium Pentosan Polysulfate (PPS) on the Differentiation ofHuman Progenitor Cells in an Adipogenic Media Using in Vitro AdipogenicAssays

The conditions required for the development of lipids from human bonemarrow stromal cells in vitro have been previously described (Gimble J.Marrow stromal adipocytes. In: Marrow stromal cell culture. J NBeresford, Owen, M. E., ed. Cambridge University Press, Cambridge, pp67-87 (1998)). Briefly, human progenitor cells were seeded into 96-wellplates at a density of 8×10³ cells per well in complete alpha-MEM growthmedia and were allowed to reach >90% confluence prior to addition ofinductive media. Cells were cultured in adipogenic-inductive mediacomprised of complete alpha-MEM supplemented with 0.5 mM3-Isobutyl-1-methyl-xanthine (IBMX), 60 microM Indomethacin, and 0.5microM Hydrocortisone in the presence of a titration of PPS (0.0, 1.0,5.0, 10 micrograms/mL). The inductive media was changed twice weekly fora period of 4 weeks. Cells were stained for the presence of lipid usingOil Red ‘O’.

Oil Red ‘O’ Staining of Lipid

Cells were cultured as described above and gently rinsed in 1× PBS(pH7.4) to avoid disruption of the cell monolayer. Cells were fixed inphosphate buffered formalin for 15 minutes at RT. The fixative wassubsequently removed and lipid stained by adding 100 microL of freshlyfiltered Oil Red O (3 mg/mL; MP Biomedicals, Australia) for ≧2 hours atRT. Cells were rinsed 3 times with RO water and counter stained withMayer's Haematoxylin (Lillie's modification). Haematoxylin stains wereaspirated and replaced by water and the Oil Red O positive adipocytesexamined under a light microscope and photographed with the DP20-56Olympus camera (Olympus, Japan).

These results are shown in FIGS. 8A and B. It can be seen that PPSregulated the differentiation of progenitor cells into adipocytes withupregulation seen at all concentrations.

Effects of Sodium Pentosan Polysulfate (PPS) on the Biosynthesis ofProteoglycans (Measured as ³⁵S-GAGS) by Progenitor Cells Grown inCollagen Sponges.

Human immunoselected Stro-1+ Progenitor cells (Mesoblast Ltd, Melbourne,Australia) prepared as a suspension containing an average of 100,000cells in 100 microL of 1:1 high glucose DMEM/Ham's F-12 medium(Invitrogen) supplemented with 10% FBS (Sigma) will be injected using amicropipett into the centre of blocks of prepared collagen spongesplaced in the wells of a 24 well culture plate. The collagen spongeswill be Sterile Gelfoams (Pharmacia & Upjohn Co., Kalamazoo, Mich.)pre-cut before hand to 0.5 cm³ cubes. To each well will be added 2 mLmedia+FBS and the plates will be incubated at 37° C., in 5% CO₂/95% airfor 48 hours. The media will then be replaced with 1:1 high glucoseDMEM/Ham's F-12 medium (Invitrogen) supplemented with 10% FBS (Sigma)containing various concentrations of PPS (0, 0.1, 0.5, 1.0, 2.5, 5.0,10.0, 25.0 μg/ml) for a further 48 hours. All PPS concentrations will becultured in triplicate. The medium will then be changed to definedmedium, which contained the indicated concentrations of PPS and 5 μCi/mlof ³⁵S-H₂SO₄ (Perkin Elmer, USA). The experiments will be terminatedafter 48 hours incubation when PG biosynthesis will be determined bymeasuring the incorporation of ³⁵SO₄ into the PG as (³⁵S-GAGs) releasedinto the media and after collagenase digestion of the sponges asdescribed above.

This experiment will demonstrate cartilage formation within the spongeis enhanced in the presence of polysulfated polysaccharides at similarconcentrations to those shown in vitro and described herein. Theexperiment will confirm that doses of 0.5-1.0 million precursor cellsare suitable cell numbers and that concentrations of 1-10 micrograms/mlpolysulfated polysaccharides provide a beneficial effect.

Evaluation of de Novo Cartilage Formulation in an Animal Model of DiscRegeneration and Cartilage Repair Using a Formulation of ProgenitorCells and Pentosan Polysulfate (PPS).

Animal Protocol

Two level spinal surgeries will be undertaken at the cervical C3/4 andC4/5 spinal levels of 12 adult Merino/Leicester ewes which will berandomly divided into 2 groups of 6. The procedure will require that theintervertebral discs be surgically removed from these levels and abiodegradable implant filled by a collagen sponge scaffold containingthe progenitor cells be implanted between the vertebral bodiespreviously occupied by the discs. Apart from the different cellsinjected into the sponges the only other variable in the design of thestudy will be whether the cartilage end plates (CEP) is mechanicallyperforated prior to insertion of the implant The duration of the studywill be 12 weeks from the time of implantation to sacrifice.

Group A (N=6)

Sterile Gelfoam Gelatin Sponges (Pharmacia & Upjohn Co., Kalamazoo,Mich., USA) will be cut to size using a preformed template, theninjected with 100 micro litres of Profreeze® solution using amicro-pipette. The loaded sponge will then be inserted into thespecially modified biodegradable cage which will be fixed within thesurgically excised cervical disc spaces at the levels indicated. Thecages will be secured in place by a means of a commercial vertebralplate.

Group B (N=6)

Sterile Gelfoam Gelatin Collagen Sponges will cut to size using apreformed template and loaded with 100 micro litres of Profreeze®solution containing progenitor cells (1 million ovine progenitorcells)+10 micrograms of PPS. The loaded sponge will then be insertedinto a biodegradable cage which will be fixed within the surgicallyexcised cervical disc spaces at the levels indicated. The cages will besecured in place by a means of a commercial vertebral plate.

Evaluation of Experimental Outcomes

Lateral radiographs will be taken of all cervical spines under inductionanaesthesia at the following time points: Baseline, Operation, 1, 2 and3 months following implantation of the test articles and scored for boneformation using the scoring system shown in Table 3.

TABLE 3 Score Description 0 no bony fusion 1 maximum intervertebral gapin the cranio-caudal direction of more than 5 mm 2 maximumintervertebral gap in the cranio-caudal direction of less than 5 mm 3complete bony fusion. The maximum intervertebral gap in thecranio-caudal direction will be measured directly on lateral radiographsusing a ruler

Animals will also be monitored throughout the study according to animalethics guidelines for care of chronically prepared sheep using theschedule shown in Table 4.

TABLE 4 Observation of animals following surgery ObservationFrequency/Day Duration Weight x1 Study entry Behaviour, Posture andActivity x1 Study duration Pain and Discomfort x1 Study durationObservation of procedural area for local x1 Minimum ofirritation/infection 3 days post surgery Decreased activity/inability tomove x1 Study duration Assessment of daily food/water x1 Study durationconsumption

Histological Analysis

Following sacrifice intact cervical spines will be dissected from theanimals and the C3/4 and C4/5 motion segments cut from the remainder ofthe spine using a band saw. These two segments will then be cut in thesagital plane into 2 sections and stored in 10% normal bufferedformalin. These sections will contain the cage with 3 mm of the superiorand inferior vertebral bodies on either side. They will then bedecalcified using formic acid and then dehydrated in ascendingconcentrations of ethanol under agitation. Following clearing in xylene,tissues will be embedded in paraffin, cut and stained with H&E, AlcianBlue, Toludine Blue, Massons Trichrome. The Toluidine Blue stainedsections will be used for the histomorphometric analysis usingquantitative image analysis to determine optical density of proteoglycandistribution and matrix dimensions using Image J® software(http://rsb.info.nih.gov/ij/) on a personal computer.

Unstained paraffin sections will also be used for immunohistochemicalanalysis of matrix components. They will be pre-digested withcombinations of chondroitinase ABC (0.25 U/ml) in 20 mM Tris-acetatebuffer pH 8.0 for 1 h at 37° C., bovine testicular hyaluronidase 1000U/ml for 1 h at 37° C. in phosphate buffer pH 5.0, followed by threewashes in 20 mM Tris-HCl pH 7.2 0.5M NaCl (TBS) or proteinase-K (DAKOS3020) for 6 min at room temperature to expose antigenic epitopes. Thetissues will then be blocked for 1 h in 20% normal swine serum andprobed with a number of primary antibodies to large and smallproteoglycans and collagens (Table 5). Negative control sections willalso be processed either omitting primary antibody or substituting anirrelevant isotype matched primary antibody for the authentic primaryantibody of interest. Commercial (DAKO) isotype matched mouse IgG (DAKOCode X931) or IgM (DAKO Code X942) control antibodies (as appropriate)will be used for this step. The DAKO products X931 and X942 will bemouse monoclonal IgG₁ (clone DAK-GO1) and monoclonal IgM (clone DAK-GO8)antibodies directed against Aspergillus niger glucose oxidase, an enzymethat is neither present nor inducible in mammalian tissues. Horseradishperoxidase or alkaline phosphatase conjugated secondary antibodies willbe used for the detection using 0.05% 3,3′-diaminobenzidenedihydrochloride and 0.03% H₂O₂ in TBS, Nova RED, nitrobluetetrazolium/5-bromo-4-chloro-3-indolylphosphate/iodonitrotetrazoliumviolet (NBT/BCIP/INT) or New Fuchsin as substrates. The stained slideswill be examined by bright field microscopy and photographed using aLeica MPS 60 photomicroscope digital camera system.

TABLE 5 Primary antibodies to proteoglycan and collagen core proteinepitopes Primary antibody epitope Clone (isotype) References LargeProteoglycans Aggrecan AD 11-2A9 (IgG) a Versican 12C5 (IgG) b CollagenType I I8H5 (IgG₁) b, c Type II II-4CII (IgG₁) b, c Type IV CIV-22(IgG₁) b, c Type VI Rabbit polyclonal b, c Type IX Mouse monoclonalsD1-9 (IgG₁), d B3-1 (IgG_(2b)) (a) Melrose, J., Little, C. B. & Ghosh,P. Detection of aggregatable proteoglycan populations by affinityblotting using biotinylated hyaluronan. Anal Biochem 256, 149-157(1998). Melrose, J., Smith, S. & Ghosh, P. Differential expression ofproteoglycan epitopes by ovine intervertebral disc cells. J Anat 197 (Pt2), 189-198 (2000). (b) Melrose, J., Smith, S., Ghosh, P. & Taylor, T.K. Differential expression of proteoglycan epitopes and growthcharacteristics of intervertebral disc cells grown in alginate beadculture. Cells Tissues Organs 168, 137-146 (2001). (c). Shen, B.,Melrose, J., Ghosh, P. & Taylor, F. Induction of matrixmetalloproteinase-2 and -3 activity in ovine nucleus pulposus cellsgrown in three-dimensional agarose gel culture by interleukin-1beta: apotential pathway of disc degeneration. Eur Spine J 12, 66-75 (2003).(d) Ye, X. J., Terato, K., Nakatani, H., Cremer, M. A. & Yoo, T. J.Monoclonal antibodies against bovine type IX collagen (LMW fragment):production, characterization, and use for immunohistochemicallocalization studies. J Histochem Cytochem 39, 265-271 (1991).

Statistics

The Student t test will be used for pair wise comparisons as indicated.Statistical significance will be given at P less than 0.05. One-wayanalysis of variance (ANOVA) will be used for multiple comparisons asindicated. Statistical significance between the groups will bedetermined using the Fisher projected least significance difference testat P less than 0.05.

This experiment will demonstrate that relative to control, the use ofpolysulfated polysaccharides and progenitor cells will result in greater(more abundant) production of cartilage in the disk space.

In addition, in the excised disc spaces with punctured cartilaginous endplates which had an interface with the collagen sponges containingprogenitor cells plus PPS, enhanced infiltration of endogenous bloodbourne progenitor cells will be observed accompanied by more completehealing of the mechanically produced cartilage defects.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

1. A composition comprising progenitor cells together with apolysulfated polysaccharide or biologically active molecular fragmentthereof.
 2. The composition of claim 1 further comprising a carriermedium.
 3. The composition of claim 2, wherein the carrier medium is aculture medium, cryopreservation medium and/or a pharmaceuticallyacceptable carrier.
 4. The composition of claim 1 wherein the progenitorcell is a chondroprogenitor cell.
 5. The composition of claim 1 whereinthe progenitor cell is a Stro-1^(bri) cell, and/or a Stro-1^(bri)progeny cell.
 6. The composition of claim 1 wherein the polysulfatedpolysaccharide is selected from pentosan polysulfate chondroitinpolysulfate, chitosan polysulfate, dextran polysulfate and heparin (highand low molecular weight) together with their pharmaceuticallyacceptable salts.
 7. The composition of claim 1 wherein the polysulfatedpolysaccharide is selected from pentosan polysulfate, the sodium salt ofpentosan polysulfate (NaPPS), the magnesium salt of pentosan polysulfate(MgPPS), and/or the calcium salt of pentosan polysulfate (CaPPS).
 8. Thecomposition of claim 1 wherein the composition comprises about 1000 toabout 1×10¹⁰ cells; about 1×10⁵-1×10⁹ cells; about 100,000 to about5×10⁸ cells; about 500,000 to about 2×10⁸ cells, about 1×10⁶ to about2×10⁸ cells; about 1×10⁶ to about 1×10⁸ cells; about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, or about 9×10⁸ cells.
 9. The composition ofclaim 1 wherein the composition comprises polysulfated polysaccharide ina concentration of about 500 ng/ml/million cells-10 mg/ml/million cells,500 ng/ml/million cells-2000 μg/ml/million cells, 1 μg/ml/millioncells-1000 μg/ml/million cells, or 1 μg/ml/million cells-500μg/ml/million cells, 500 ng-10 μg/ml/million cells, 1 μg-10μg/ml/million cells, 1 μg-8 μg/ml/million cells, 1 μg-6 μg/ml/millioncells, 1 μg-5 μg/ml/million cells, 1 μg-3 μg/ml/million cells, 2 μg-6μg/ml/million cells, 2.5 μg-5 μg/ml/million cells, or 3 μg-5μg/ml/million cells, 1 μg-100 μg/ml/million cells, 1 μg-50 μg/ml/millioncells, 1 μg-20 μg/ml/million cells, 1 μg-15 μg/ml/million cells, 10μg-100 μg/ml/million cells, 20 μg-100 μg/ml/million cells, or 50 μg-100μg/ml/million cells, 1 μg-1000 μg/ml/million cells, 100 μg-800μg/ml/million cells, 100 μg-600 μg/ml/million cells, 100 μg-500μg/ml/million cells, 200 μg-500 μg/ml/million cells, 500 ng, 1 μg, 2 μg,2.5 μg, 5 μg, 10 μg, 15 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80μg, 90 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900μg, 950 μg, 1000 μg, 1050 μg, 1100 μg, 1150 μg, 1200 μg, 1250 μg, 1300μg, 1350 μg, 1400 μg, 1450 μg, 1500 μg, 1550 μg, 1600 μg, 1650 μg, 1700μg, 1750 μg, 1800 μg, 1850 μg, 1900 μg, 1950 μg, or 2000 μg/ml/millioncells.
 10. The composition of claim 1 wherein the composition comprisesabout 1×10⁶-1×10⁸ progenitor cells and 25-50 mg polysulfatedpolysaccharide; 1×10⁸ progenitor cells and 25-50 mg/ml polysulfatedpolysaccharide.
 11. The composition of claim 1 wherein the compositionfurther comprises one or more of the following components: organicand/or inorganic salts; buffers proteins such as BSA or transferin;growth factors and cytokines, including insulin like growth factor,insulin, fibroblast like growth factors; BMP-TGF-beta super family (eg,BMP-2, BMP-7, BMP-8, TGF beta) and fibroblast growth factor family, IGF,FGF, EGF, PDGF, VEGF; animal sera including FBS, new-born calf, allother mammalian species; cryopreservation agents such as Profreeze® andCryoStor®; cryoprotectorants, including dimethyl sulfoxide (DMSO),glycerol, trehalose, sucrose and other sugars or dimethylacetamide;carbohydrates; vitamins/co-factors; hormones antibiotics attachmentfactors; amino acids; plasma expanders like dextran; plasma both humanand other mammalian species; plasma substitute; hyaluronan and/orhyaluronic acid, both natural or cross linked.
 12. The composition ofclaim 11 wherein the DMSO is present in the range of 1-20%, 1-15%,5-15%, 1-10%, 5%, 7.5%, 10%, 15% or 20%; and/or the FBS is in the rangeof 1-50%, 1-20%, 1-10%, 5%, 7.5%, 10%, 15% or 20%.
 13. The compositionof claim 1 wherein the carrier medium comprises Profreeze®.
 14. Thecomposition of claim 1 wherein the composition further comprises NC4 ora biologically active fragment thereof.
 15. A method of enhancingcryopreservation of progenitor cells, comprising exposing the progenitorcells to a polysulfated polysaccharide or a biologically activemolecular fragment thereof. 16-17. (canceled)
 18. A method of regulatingthe proliferation of progenitor cells, comprising exposing apolysulfated polysaccharide or biologically active molecular fragmentthereof to a progenitor cell.
 19. (canceled)
 20. A method of regulatingdifferentiation of progenitor cells by exposing a polysulfatedpolysaccharide or biologically active molecular fragment thereof to aprogenitor cell, including differentiation into chondrocytes,differentiation into osteoblasts or differentiation into adipocytes.21-22. (canceled)